Malleable hydrogel hybrids made of self-assembled peptides and biocompatible polymers and uses thereof

ABSTRACT

Hybrid hydrogels formed of a plurality of peptides that are capable of self-assembling into a hydrogel in an aqueous solution and a biocompatible polymer that is characterized by high swelling capability, high elasticity and low mechanical strength are disclosed, with exemplary hybrid hydrogels being formed of a plurality of aromatic dipeptides and hyaluronic acid. The hybrid hydrogels are characterized by controllable mechanical and biological properties which can be adjusted by controlling the concentration ratio of the peptides and the polymer, and which average the mechanical and biological properties of the peptides and the polymer. Processes of preparing the hydrogels and uses thereof in pharmaceutical, cosmetic or cosmeceutic applications such as tissue engineering and/or regeneration are further disclosed.

RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.13/701,558 filed on Dec. 3, 2012, which is a National Phase of PCTPatent Application No. PCT/IL2011/000435 having International filingdate of Jun. 2, 2011, which claims the benefit of priority under 35 USC§119(e) of U.S. Provisional Patent Application No. 61/350,978 filed onJun. 3, 2010. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to novelbiomaterials and, more particularly, but not exclusively, to malleablehybrid hydrogels made of peptide(s) and polymer(s).

Biomaterials for tissue engineering are required to possess uniquecharacteristic features for allowing proper biocompatibility, supportinggrowth and handy biomechanical properties enabling injectability andmalleability of the matrix substances.

Development of malleable polymeric nanofiber constructs is of a greatscientific and technological interest due to their wide-rangeapplications in biomedicine and biotechnology. Particularly, compositenanofibers derived from natural and synthetic polymers, combining thefavorable biological properties of the natural polymer and themechanical strength of the synthetic polymer, represents a majoradvantageous advancement in tissue engineering and regenerativemedicine.

Hydrogels are determined as polymer networks that are insoluble inwater, where they swell to an equilibrium volume but retain theirshapes. Hydrogels are of great interest as a class of materials fortissue engineering and regenerative medicine, as they offer 3D scaffoldsto support the growth of cultured cells. In terms of materialrequirements, hydrogels have long received attention because of theirinnate structural and compositional similarities to the extracellularmatrix and their extensive framework for cellular proliferation andsurvival.

A variety of natural polymers, including agarose, collagen, fibrin,alginate, gelatin, chitosan and hyaluronic acid (HA), may be used ashydrogel-forming materials [Almany and Seliktar 2005, Biomaterials 26:2467-2477]. These polymers are appealing for medical use owing to theirsimilarity to the natural extracellular matrix (ECM), which allows celladhesion, migration and proliferation, while maintaining very goodbiocompatible and biodegradable qualities.

Another class of building blocks for hydrogel formation includessynthetic materials, such as poly(ethylene oxide), poly(vinyl alcohol)and poly[furmarate-co-(ethylene glycol)]. These synthetic buildingblocks offer controllability and reproducibility, but several drawbacksinclude their production methods that sometimes involve the use ofextreme temperatures and pressures and the use of complex techniques, aswell as low biocompatibility of the products.

Hydrogels can be prepared either by production of chemical gels or byproduction of physical gels. Chemical hydrogels are produced bycrosslinking starting materials, through chemical or polymerizationreactions, and hence involve covalent linking of the hydrogel networks.Physical hydrogels are receiving a great attention since theirproduction does not involve chemical reactions, a fact which isadvantageous in the context of encapsulation of cells and othersensitive molecules therein, since laborious removal of toxic orextremely reactive molecules used to initiate chemical crosslinkingreactions is circumvented. In physical hydrogels, the networks are heldtogether by molecular entanglements, and/or secondary forces includingelectrostatic forces, hydrogen-bonding forces or hydrophobic forces[Campoccia et al. 1998 Biomaterials 19: 2101-2127; Prestwich et al. 1998J. Controlled Release 53: 93-103].

Hyaluronic acid (HA) is a high molecular weight unsulfatedglycosaminoglycan (GAG) present in all mammals. HA is composed ofrepeating disaccharide units composed of (β-1,4)-linked D-glucuronicacid and (β-1,3)-linked N-acetyl-D-glucosamine (see, FIG. 1A). GAG, amajor component of the native extracellular matrix (ECM), is known tosupport enhanced cell attachment and proliferation and to improve thematerial's cellular and tissue biocompatibilities. HA in the body occursas its sodium salt form hyaluronate and is found in high concentrationsin the fetus, umbilical cord, and in several soft connective tissues ofadults, including skin, synovial fluid, and vitreous humor.

Being a highly hydrated, negatively charged, linear biodegradable andbiocompatible natural polymer, characterized by high viscoelastic andspace filling properties, HA is highly useful for tissue engineeringapplications. The advantageous rheological features of HA are exploited,for example, in the application of hyaluronan for ophthalmic surgery[Pape and Balazs 1980, Ophthalmology 87(7): 699-705.], in the cosmeticfield [Duranti et al 1998 Dermatol. Slug. 24: 1317-1325], and in theintra-articular treatment of osteoarthritis [Goa and Benfield 1994 Drugs47(3): 536-566].

However, the use of HA is limited by its poor mechanical strength and byits rapid in vivo enzymatic digestion by hyaluronidase. Overcoming theselimitations can be made by introducing synthetic cross-linkers, forproviding strengthen HA composite with reduced biodegradation rate[Leach et al. 2003 Biotechnol. Bioeng. 82(5), 578-589; Lu et al. 2008 J.Biomater. Sci. Polym. 19:1-18; and Pitarresi et al. 2008 J. Biomed.Mater. Res, Part A 84A(2): 413-424], and/or by employing specific ornon-specific inhibitors of hyaluronidase.

Self-assembled nanotubes and hydrogels made of short (aromatic) peptideshave been disclosed in Mahler et al. Adv. Mater. 18: 1365-1370, and inWO 2007/0403048, WO 2004/052773 and WO 2004/060791. An exemplarybuilding block for forming such nanotubes and hydrogels is Fmoc-FF.

Fmoc-FF is a protected dipeptide, which was shown to self-assemble intodiscrete, well-ordered nanotubes and to form hydrogels in themacrostructure. The diphenylalanine peptide (FF) is the natural corerecognition motif of the amyloid-βpolypeptide. The Fmoc group(9-fluorenylmethoxycarbonyl) is widely used as a synthetic protectinggroup in peptide synthesis and it was reported by Burch et al. that anumber of Fmoc-amino acids show anti-inflammatory properties [Burch etal. 1991 Proc. Natl. Acad. Sci. U.S.A. 88: 355-359].

The efficient self-assembly, under mild conditions, of Fmoc-FF into ahydrogel which exhibits remarkable physical properties has been reported(see, Mahler et al., supra). In spite of the short building-block size,the obtained hydrogel was characterized by physical properties thatexceed those of hydrogels formed from longer polypeptides.

Hybrid composite hydrogels are reviewed, for example, in Jia and Kiickin Macromol Biosci. 2009 February 11; 9(2): 140-156, and referencescited therein. Several hybrid hydrogels made of hyaluronic acid andpolymers such as PEG, chitosan, cellulose and alginate have beenreported. Hybrid hydrogels made of hyaluronic acid and otherpolysaccharides and proteins such as collagen, gelatin and fibrin havealso been reported. Hydrogel matrices made of hyaluronic acidderivatized by a cell adhesive peptide fragment are disclosed, forexample, in U.S. Pat. Nos. 5,834,029 and 6,156,572. Hydrogels made ofhyaluronic acid modified with Nodo-66 antagonist have been reported inHou et al., J. Neurosci. Met. 137:519-529, 2005.

Additional background art includes Yang et al., Biomedical Materials,2001, 6, 025009; Kim et al., Acta Biomater. 2008, 4(6):1611-1619; andPark et al., Key engineering materials, Vols. 342-343 (2007), pp.153-156.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a hydrogel comprising a fibrous network of a pluralityof peptides and hyaluronic acid, wherein each peptide in the pluralityof peptides is a dipeptide which comprises at least one aromatic aminoacid residue.

According to some embodiments of the invention, at least one of thedipeptides consists of aromatic amino acid residues.

According to some embodiments of the invention, each of the dipeptidesconsists of aromatic amino acid residues.

According to some embodiments of the invention, at least one dipeptidein the plurality of dipeptides is a homodipeptide.

According to some embodiments of the invention, each of the dipeptidesis a homopeptide.

According to some embodiments of the invention, each of the dipeptidesis phenylalanine-phenylalanine dipeptide (Phe-Phe). According to someembodiments of the invention, at least one dipeptide in the plurality ofpeptides is an end-capping modified peptide.

According to some embodiments of the invention, each dipeptide in theplurality of dipeptides is an end-capping modified peptide.

According to some embodiments of the invention, each of the end-cappingmodified dipeptides comprises an aromatic end-capping moiety.

According to some embodiments of the invention, the aromatic end cappingmoiety is 9-fluorenylmethyloxycarbonyl (Fmoc).

According to an aspect of some embodiments of the invention there isprovided a hydrogel comprising a fibrous network of a plurality ofpeptides and at least one biocompatible polymer, wherein the peptidesare capable of self-assembling in an aqueous solution so as to form ahydrogel and wherein the biocompatible polymer features at least onecharacteristic selected from the group consisting of: (i) a storagemodulus G′ lower than 500 Pa at 10 Hz frequency and at 25° C.; (ii) aswelling ratio (Q) higher than 500; (iii) a viscosity at 0.1 Sec⁻¹shearrate and at 25° C., lower than 300 Pa·s; and (iv) a viscosity recoveryafter shear of at least 95%.

According to some embodiments of the invention, the biocompatiblepolymer is a polysaccharide.

According to some embodiments of the invention, the biocompatiblepolymer is hyaluronic acid.

According to some embodiments of the invention, the biocompatiblepolymer is a chitosan.

According to some embodiments of the invention, the biocompatiblepolymer has an average molecular weight that ranges from 10 kDa to10,000 kDa.

According to some embodiments of the invention, each peptide in theplurality of peptides comprises an amino acid sequence not exceeding 6amino acids in length, whereas the amino acid sequence comprises atleast one aromatic amino acid residue.

According to some embodiments of the invention, at least one peptide inthe plurality of peptides consists essentially of aromatic amino acidresidues.

According to some embodiments of the invention, each peptide in theplurality of peptides consists essentially of aromatic amino acidresidues.

According to some embodiments of the invention, at least one peptide inthe plurality of peptides is a dipeptide.

According to some embodiments of the invention, each peptide in theplurality of peptides is a dipeptide.

According to some embodiments of the invention, at least one of thedipeptides is a homodipeptide.

According to some embodiments of the invention, each of the dipeptidesis a homopeptide.

According to some embodiments of the invention, the homodipeptide isselected from the group consisting of phenylalanine-phenylalaninedipeptide, naphthylalanine-naphthylalanine dipeptide,phenanthrenylalanine-phenanthrenylalanine dipeptide,anthracenylalanine-anthracenylalanine dipeptide,[1,10]phenanthrolinylalanine-[1,10]phenanthrolinylalanine dipeptide,[2,2′]Thipyridinylalanine42,2Thipyridinylalanine dipeptide,(pentahalo-phenylalanine)-(pentahalo-phenylalanine) dipeptide,(amino-phenylalanine)-(amino-phenylalanine) dipeptide,(dialkylamino-phenylalanine)-(dialkylamino-phenylalanine) dipeptide,(halophenylalanine)-(halophenylalanine) dipeptide,(alkoxy-phenylalanine)-(alkoxy-phenylalanine) dipeptide,(trihalomethyl-phenylalanine)-(trihalomethyl-phenylalanine) dipeptide,(4-phenyl-phenylalanine)-(4-phenyl-phenylalanine) dipeptide and(nitro-phenylalanine)-(nitro-phenylalanine) dipeptide.

According to some embodiments of the invention, at least one peptide inthe plurality of peptides is an end-capping modified peptide.

According to some embodiments of the invention, each peptide in theplurality of peptides is an end-capping modified peptide.

According to some embodiments of the invention, the end capping modifiedpeptide comprises at least one end capping moiety, the end cappingmoiety being selected from the group consisting of an aromatic endcapping moiety and a non-aromatic end-capping moiety.

According to some embodiments of the invention, the aromatic end cappingmoiety is 9-fluorenylmethyloxycarbonyl (Fmoc).

According to some embodiments of the invention, a total concentration ofthe plurality of peptides and the polymer ranges from 0.1 weight percentto 5 weight percents of the total weight of the gel.

According to some embodiments of the invention, the total concentrationranges from 0.5 weight percent to 2.5 weight percent of the total weightof the gel.

According to some embodiments of the invention, a weight ratio of thedipeptides and the polymer ranges from 10:1 to 1:10.

According to some embodiments of the invention, the ratio ranges from3:1 to 1:3.

According to some embodiments of the invention, the fibrous networkcomprises a plurality of fibrils, whereas an average diameter of thefibrils ranges from about 10 nm to about 100 nm.

According to some embodiments of the invention, the hydrogel ischaracterized by a storage modulus G′ to loss modulus G″ ratio that ishigher by at least 2-folds than the ratio of the biocompatible polymer.

According to some embodiments of the invention, the hydrogel ischaracterized by a storage modulus G′ that is lower by at least 10% of astorage modulus G′ of a hydrogel formed of the plurality of peptides.

According to some embodiments of the invention, the hydrogel ischaracterized by a storage modulus G′ that is higher by at least 5-foldsof a storage modulus G′ of the biocompatible polymer.

According to some embodiments of the invention, the hydrogel ischaracterized by a swelling ratio (Q) higher by at least 5% of aswelling ratio of a hydrogel formed of the plurality of peptides.

According to some embodiments of the invention, the hydrogel ischaracterized by a viscosity higher by at least 10% of a viscosity ofthe biocompatible polymer.

According to some embodiments of the invention, the hydrogel ischaracterized by a viscosity change through time higher by at least2-folds than a viscosity change through time of the biocompatiblepolymer.

According to some embodiments of the invention, the hydrogel ischaracterized by biocompatibility to cell viability higher by at least2-folds than a biocompatibility to cell viability of a hydrogel formedof the plurality of peptides.

According to some embodiments of the invention, the hydrogel ischaracterized by a storage modulus G′ to loss modulus G″ ratio that isgreater than 4.

According to some embodiments of the invention, the hydrogel ischaracterized by a storage modulus G′ higher than 1,000 Pa at 10 Hzfrequency and at 25° C.

According to some embodiments of the invention, the hydrogel ischaracterized by a storage modulus G′ lower than 100,000 Pa at 10 Hzfrequency and at 25° C.

According to some embodiments of the invention, the hydrogel ischaracterized by a viscosity that ranges from 200 to 2000 Pa·s at 0.1Sec⁻¹ shear rate, at 25° C.

According to some embodiments of the invention, the hydrogel ischaracterized by a viscosity recovery after shear of at least 50%, at0.1 sec⁻¹.

According to some embodiments of the invention, the hydrogel ischaracterized by a swelling ratio (Q) that ranges from 100 to 500.

According to an aspect of some embodiments of the invention there isprovided a composition-of-matter comprising the hydrogel as describedherein and at least one agent being incorporated therein or thereon.

According to some embodiments of the invention, the agent is selectedfrom the group consisting of a therapeutically active agent, adiagnostic agent, a biological substance and a labeling moiety.

According to some embodiments of the invention, the agent is selectedfrom the group consisting of a drug, a cell, a nucleic acid, afluorescence compound or moiety, a phosphorescence compound or moiety, aprotein, an enzyme, a hormone, a growth factor, a bacterium and aradioactive compound or moiety.

According to an aspect of some embodiments of the invention there isprovided a process of preparing the hydrogel as described herein, theprocess comprising contacting the plurality of peptides and thebiocompatible polymer in an aqueous solution.

According to some embodiments of the invention, the contacting comprisesdissolving the polymer in the aqueous solution and contacting thepeptides with the aqueous solution.

According to some embodiments of the invention, a total concentration ofthe plurality of peptides and the polymer in the aqueous solution rangesfrom about 0.1 mg/ml to about 50 mg/ml.

According to some embodiments of the invention, the process furthercomprises, prior to the contacting, dissolving the plurality of peptidesin a water-miscible organic solvent.

According to some embodiments of the invention, the contacting iseffected ex-vivo.

According to some embodiments of the invention, the contacting iseffected in-vivo.

According to some embodiments of the invention, the contacting iseffected at a desired site of application of the hydrogel.

According to an aspect of some embodiments of the invention there isprovided a pharmaceutical, cosmetic or cosmeceutical compositioncomprising the hydrogel of as described herein.

According to some embodiments of the invention, the composition furthercomprises a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the invention there isprovided a pharmaceutical, cosmetic or cosmeceutical compositioncomprising the composition-of-matter as described herein.

According to an aspect of some embodiments of the invention there isprovided an article-of-manufacture comprising the hydrogel,composition-of-matter or composition as described herein.

According to an aspect of some embodiments of the invention there isprovided a kit for forming the hydrogel as described herein, the kitcomprising the plurality of peptides and the biocompatible polymer.

According to some embodiments of the invention, the kit furthercomprises instructions for forming the hydrogel by contacting thepeptides and the biocompatible polymer with an aqueous solution.

According to some embodiments of the invention, the kit furthercomprises an aqueous solution, wherein the peptides and the aqueoussolution are individually packaged within the kit.

According to some embodiments of the invention, the polymer is dissolvedin the aqueous solution.

According to an aspect of some embodiments of the invention there isprovided a kit of for forming the composition-of-matter as describedherein, the kit comprising the plurality of peptides, the biocompatiblepolymer and the active agent.

According to some embodiments of the invention, the kit furthercomprises instructions for forming the hydrogel by contacting theplurality of peptides, the biocompatible polymer and the active agentwith an aqueous solution.

According to some embodiments of the invention, the kit furthercomprises an aqueous solution, wherein the plurality of peptides and theaqueous solution are individually packaged within the kit.

According to some embodiments of the invention, the polymer is dissolvedin the aqueous solution.

According to some embodiments of the invention, each of the hydrogel,the composition-of-matter or the composition described herein isidentified for use in repairing a damaged tissue.

According to an aspect of some embodiments of the invention there isprovided a use of the hydrogel, the composition-of-matter or thecomposition as described herein in the manufacturing of a medicament forrepairing a damaged tissue.

According to an aspect of some embodiments of the invention there isprovided a method of repairing a damaged tissue, the method comprisingcontacting the damaged tissue with the hydrogel, thecomposition-of-matter or the composition as described herein.

According to an aspect of some embodiments of the invention there isprovided a method of repairing a damaged tissue comprising contactingthe damaged tissue with a mixture comprising a plurality of peptides anda biocompatible polymer, wherein the peptides are capable ofself-assembling in an aqueous solution so as to form a hydrogel andwherein the biocompatible polymer features at least one characteristicas described herein.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-B present the chemical structures of hyaluronic acid (HA, FIG.1A) and Fmoc-diphenylalanine (Fmoc-FF, FIG. 1B), exemplary buildingblock of the hybrid hydrogels according to some embodiments of thepresent invention;

FIG. 2 is a photograph showing the hydrogel macrostructure of hydrogelsmade of pure components (DDW, Fmoc-FF and HA) and of hybrid hydrogelsmade from different weight ratios of Fmoc-FF/HA;

FIGS. 3A-C present images showing the hydrogel microstructure ofhydrogels made of HA (1), 25/75 Fmoc-FF/HA (2), 50/50 Fmoc-FF/HA (3),75/25 Fmoc-FF/HA (4) and Fmoc-FF (5), as observed by TEM (FIG. 3A), SEM(FIG. 3B) and E-SEM (FIG. 3C);

FIG. 4 is a bar graph presenting the density values calculated forsolutions (DDW and PBS) and hydrogels made of pure components (Fmoc-FFand HA) and of hybrid hydrogels made from different weight ratios ofFmoc-FF/HA (n=3-6);

FIG. 5 is a bar graph presenting the swelling ratio calculated forhydrogels made of pure components (Fmoc-FF and HA) and of hybridhydrogels made from different weight ratios of Fmoc-FF/HA, as an averageof values obtained for 3 hydrogels of each hydrogel type at 4 timepoints during 2 weeks;

FIG. 6 presents comparative plots showing the viscosity at roomtemperature of hydrogels made of HA (1%; black Xs), 25/75 Fmoc-FF/HA(0.5%; red squares), 75/25 Fmoc-FF/HA (0.5%; blue diamonds), 50/50Fmoc-FF/HA (black triangles) and Fmoc-FF (0.5%, black +);

FIG. 7 presents comparative plots showing the values obtained in anelasticity test measuring the viscosity recovery after shear of a 50/50Fmoc-FF/HA hybrid hydrogel and of a Fmoc-FF hydrogel, with blacktriangles representing data for Fmoc-FF hydrogel at t=0, blank trianglesof Fmoc-FF hydrogel at t=10, black circles representing data for a 50/50Fmoc-FF/HA hybrid hydrogel at t=0 and blank circles of 50/50 Fmoc-FF/HAat t=10;

FIG. 8 presents comparative plots showing the rheological properties(measured at 25 ° C.) of hydrogels made of the pure components Fmoc-FF(red) and HA (blue) and of hybrid hydrogels made from 25/75 Fmoc-FF/HA(green), 75/25 Fmoc-FF/HA (cyan Xs) and 50/50 Fmoc-FF/HA (purple);

FIG. 9 presents comparative plots showing the rheological properties(measured at 25 ° C.) of hydrogels made of 75/25 Fmoc-FF/HA at aconcentrations of 0.5% (green triangles) and 1% (purple triangles) andof 50/50 Fmoc-FF/HA at a concentration of 0.5% (blue circles) and 1%(orange circles);

FIG. 10 presents comparative plots showing the effect of the temperatureon the rheological properties of an exemplary hybrid hydrogel made from50/50 Fmoc-FF/HA at 4° C. (blue), 25° C. (red) and 37° C. (black);

FIG. 11 presents comparative plots showing the hydrogel mass loss withtime for solutions made of HA in the absence (filled black squares) andpresence (blank black squares) of Hyaluronidase, and for hybridhydrogels made of 25/75 Fmoc-FF/HA in the absence (filled red diamonds)and presence (blank red diamonds) of Hyaluronidase, and of 50/50Fmoc-FF/HA in the absence (filled green triangles) and presence (blankgreen triangles) of Hyaluronidase;

FIG. 12 presents comparative plots showing release of glucuronic acidwith time from solutions made of HA in the absence (blue diamonds) andpresence (red squares) of Hyaluronidase, from hydrogels made of 25/75Fmoc-FF/HA in the absence (orange circles) and presence (purple lines)of Hyaluronidase, and from hydrogels made of 50/50 Fmoc-FF/HA in theabsence (green triangles) and presence (gray Xs) of Hyaluronidase;

FIG. 13 is a bar graph showing CHO (Chinese Hamster Ovaries) cellsviability 1 day (blue), 3 days (red) and 7 days (green) post seeding thecell on a 50/50 Fmoc-FF/HA exemplary hydrogel according to someembodiments of the invention;

FIG. 14 presents a light micrograph of chondrocytes grown on a hybridhydrogel made of 50/50 Fmoc-FF/HA, one day post seeding;

FIGS. 15A-B are bar graphs presenting the percents of cells found inmedium and gel out of the total cell amount at different time points(FIG. 15A), and the percents of cells found grown on the plate out ofthe total cell amount at different time points (FIG. 15B), upon seedingcells on top of an HA solution, a 25/75 Fmoc-FF/HA hydrogel and a 50/50Fmoc-FF/HA, as measured 2 days (blue), 6 days (red) and 10 days (green)post seeding; and

FIGS. 16A-C are light micrographs of H&E staining of limb-buds embeddedin 50/50 Fmoc-FF/HA hybrid hydrogel after 6 days on a CAM of an 8-dayfertilized chicken egg (X 25), presenting an initial stage of limb-bud(FIG. 16A), chondrocytes condensation (FIG. 16B), and organogenesis intolimb formation (FIG. 16C).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to novelbiomaterials and, more particularly, but not exclusively, to malleablehybrid hydrogels made of peptides and polymers.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples.

In view of the recognized need for mimics of extracellular matrix (ECM)in biomaterial applications such as tissue engineering, extensiveefforts have been made for designing structures that mimic the hybridnature of the natural ECM. To this effect, multicomponent hybridhydrogels have been developed by integrating modular and heterogeneousbuilding blocks into multifunctional hydrogel composites.

As further discussed hereinabove, hydrogels are the most appealingcandidates for tissue engineering scaffolds due to their structuralsimilarity to the natural ECM, inherent biocompatibility, tunableviscoelasticity, high water content and high permeability for oxygen andessential nutrients.

Hyaluronic acid represents a desirable component for hydrogel formationdue to its exceptional biocompatibility, swelling capability andviscoelasticity. The fast biodegradation of HA, however, limits its usein tissue engineering applications. Similar properties are exhibitedalso by other polysaccharides, as is further detailed hereinbelow.

Hydrogels made of self-assembled peptides have been reported, forexample, in Mahler et al. Adv. Mater. 18: 1365-1370, and in WO2007/0403048. Exemplary such hydrogels, formed from Fmoc-FF, were shownto exhibit exceptional mechanical strength. Such a high rigidity isoften not desired in tissue engineering or regeneration applications,which require malleability, and further, render such hydrogels lesssuitable matrices for maintaining or promoting cellular activity, asfurther defined hereinbelow.

In a search for improved hydrogel materials for use in biomaterialapplications such as tissue engineering and regeneration, the presentinventors have designed and successfully practiced a novel approach forcreating hybrid hydrogel composites which combine the properties ofbiocompatible polymers that are suitable matrices for cell growth withthe mechanical properties of self-assembled peptides, whilecircumventing the use of chemical crosslinking.

While conceiving the present invention, it was envisioned that combiningthe beneficial properties of biocompatible polymers that have highswelling capability and viscoelasticity with the mechanical strengthimparted by self-assembled peptides would result in composite substancesthat integrate these beneficial properties.

While reducing the present invention to practice, the present inventorshave indeed demonstrated that mixing such components under conditionsthat facilitate hydrogel formation (e.g., at suitable concentrations inan aqueous solution), without using any chemical crosslinking agents,results in hydrogels that are characterized both by a remarkablerigidity and biocompatibility. The present inventors have surprisinglyuncovered that the obtained hybrid hydrogels exhibit averaged propertiesof the two components, such that the rheological, viscoelastic, swellingand biodegradability properties of the hybrid hydrogel can be finelytuned by means of varying the concentration ratio of the two components.

The present inventors have demonstrated that well-blended compositehydrogels that integrate the favorable biological properties ofbiocompatible polymers and the mechanical properties of self-assembledpeptides significantly improve the material properties, while providinga stable, nurturing environment for a broad array of biomedicalapplications.

This novel approach was shown to improve the mechanical properties ofbiocompatible polymers such as hyaluronic acid, which are otherwisecharacterized by limited mechanical strength and sometimes by fastdegradation, and on the same time, to improve the biocompatibility ofself-assembled peptidic hydrogels, by reducing the rigidity andenhancing the swelling capability thereof, and thus by renderingmalleable hydrogels and more suitable matrices for cell growth.

As demonstrated in the Examples section that follows, hybrid hydrogelscomprised of hyaluronic acid (HA; see, FIG. 1A) as an exemplarybiocompatible polymer and Fmoc-FF(fluorenylmethoxycarbonyl-diphenylalanine; see, FIG. 1B) as an exemplarya self-assembled peptide were prepared and characterized.

HA is a biodegradable, non-immunogenic, and biocompatible naturalpolymer, which represents remarkable viscoelastic properties, and is anattractive biomaterial for cells in tissue engineering. Fmoc-FF is ashort peptide with a protected group (Fmoc), which was shown toself-assemble into remarkably rigid hydrogel microstructure.

A set of hybrid hydrogels with varying concentration ratios between thetwo components was successfully prepared and was shown to exhibit acontrollable malleability. Thus, for example, it was shown that inhigher peptide concentrations less swelling is attained, and thehydrogel represents more dense and rigid features, contributing toslower biodegradation. The hydrogel hybrids exhibit desirable mechanicalproperties (rheological-shear stress, viscosity, recovery after shear)with high biocompatibility for various cells. It has been shown that HAimproves the hydrogel hybrid elasticity, and contributes to celladhesion and biocompatibility, whereas Fmoc-FF improves the hydrogelhybrid mechanical features (shear stress), and slowing down the HAdegradation (probably due to less penetration of the degrading enzyme).

Thus, it has been shown that the blended hybrid reflects moderate,averagable biomechanical features, ranging in between the pure elements.Hybrid hydrogels were also successfully prepared from Fmoc-FF andchitosan.

According to an aspect of some embodiments of the present inventionthere is provided a hydrogel comprising a fibrous network of a pluralityof peptides and a biocompatible polymer.

In some embodiments, the plurality of peptides comprises peptides whichare capable of self-assembling in an aqueous solution so as to form ahydrogel.

As noted hereinabove, self-assembling peptides typically form relativelyrigid structures which render hydrogels formed therefrom less suitablefor biomaterial applications that typically require malleable matricessuitable for cell growth, for transport of biological substance.

As further discussed hereinabove, it has been demonstrated herein thathydrogels made of self-assembling peptides can be rendered suitable forbiomaterial applications upon forming a hybrid hydrogel which comprisessuch peptides and a polymer that imparts biocompatibility to thehydrogel by featuring characteristics such as elasticity, high swellingcapability and/or lower mechanical strength compared to theself-assembling peptides.

To this end, biocompatible polymers suitable for use within thehydrogels described herein are selected as featuring one or more of thefollowing characteristics:

(i) a storage modulus G′ lower than 500 Pa at 10 Hz frequency and at 25°C., which is indicative of a polymer with relatively low mechanicalstrength;

(ii) a swelling ratio (Q) higher than 500, which is indicative of highhydration capability of the polymer;

(iii) a viscosity at 0.1 Sec ⁻¹ shear rate and at 25° C., lower than 300Pa·s; and

(iv) a viscosity recovery after shear of at least 95%, which isindicative of elasticity.

As used herein, and is well-known in the art, the term “hydrogel” refersto a material that comprises solid, typically fibrous networks formed,at least in part, of water-soluble natural or synthetic polymer chains,and typically containing more than 90% water, or more than 95% water.

As used herein the phrase “fibrous network” refers to a set ofconnections formed between the plurality of fibrous components. Herein,the fibrous components are composed, at least in part, of peptidefibrils, each formed upon self-assembly of short peptide buildingblocks, as is further detailed hereinbelow.

Since the hydrogels described herein are formed of two or more types ofcomponents, the hydrogels are also referred to herein interchangeably as“hydrogel hybrid” or “hybrid hydrogel” or “composite” or “hydrogelcomposite” or “hybrid hydrogel composite” or “hydrogel hybridcomposite”.

As currently accepted in the art, the term “biocompatible” is generallydefined as “the ability of a material to perform with an appropriatehost response in a specific application” [see, The Williams dictionaryof Biomaterials].

In the context of biomaterial applications such as tissue engineeringand regeneration, biocompatibility refers to the ability to perform as asupportive matrix to an appropriate cellular activity, without elicitingany undesirable effects in those cells, or inducing any undesirablelocal or systemic responses in the host.

In the context of embodiments of the present invention, a “biocompatiblematerial” describes a material (e.g., a natural or synthetic polymer) ormatrix (e.g., hydrogel or scaffold) that does not interfere, andpreferably provides a suitable environment for, cellular activity.

A “cellular activity” includes, for example, cell viability, cell growth(proliferation), cell differentiation, cell migration, cell adhesion,molecular and mechanical signaling systems, and fluid transport throughcells or a tissue so as to allow nutritive environment.

The biocompatibility of a substance can be determined by methods wellknown in the art, following the definitions hereinabove andinternational guidelines, using widely recognized safety assays.Optionally, biocompatible substances can be selected from existing listsof such substances.

In the context of the present embodiments, the biocompatible polymerfeatures physical properties as described herein.

Thus, in some embodiments, the biocompatible polymer is characterized bya relatively high swelling ratio.

As used herein and in the art, the phrase “swelling ratio”, denoted “Q”,describes, the ratio between the weight of a swollen substance (Ws) andthe weight of dry substance (Wd) and is calculated according to theexpression: Q=Ws/Wd.

An exemplary procedure for measuring the weights of swollen and drysubstances in presented in the Examples section that follows.

Substances that have high swelling capability typically have a swellingratio that is higher than 100, higher than 200, higher than 300, higherthan 400, preferably higher than 500, higher than 600, higher than 700,higher than 800, higher than 900 and even 1,000.

In the context of embodiments of the invention, a high swelling ratio ofthe polymer is desired for imparting swelling capability to the formedhybrid hydrogel.

Further in the context of embodiments of the present invention,hydrogels with enhanced swelling capability are beneficial inbiomaterial applications.

Biocompatible polymers with high swelling ratio (e.g., 500 or higher)are typically highly-hydrated polymers. Exemplary such polymers includepolysaccharides, particularly high molecular weight linearpolysaccharides such as the GAGs, chitosan, agarose, alginate and thelike.

In some embodiments, the biocompatible polymer is characterized as aviscoelastic substance.

Viscoelasticity is the property of materials that exhibit both viscousand elastic characteristics when undergoing deformation. Viscousmaterials resist shear flow and strain linearly with time when a stressis applied. Elastic materials strain instantaneously when stretched andreturn to their original state once the stress is removed. Viscoelasticmaterials have elements of both of these properties and, as such,exhibit time dependent strain.

Viscoelastic substances have an elastic component and a viscouscomponent. The rheology of viscoelastic substances is thereforetypically defined by the Complex Dynamic modulus, G, which representsthe relation between the oscillating stress and strain, as follows:

G=G′+iG″

where i²=−1; G′ is the storage modulus (representing elastic modulus)and G″ is the loss modulus (representing frictional modulus).

Since it is desirable that the biocompatible polymer would impartelasticity to the formed hydrogel hybrid, is some embodiments, thebiocompatible polymer is characterized by relatively low storage modulusG′, being lower than 500, lower than 400, lower than 300, or lower than200.

In some embodiments, a biocompatible polymer is characterized by arelatively low ratio of shear storage modulus to loss modulus. In someembodiments, this ratio is lower than 1.

In some embodiments, the elasticity of the biocompatible polymer isdetermined by a change of viscosity through time and/or by viscosityrecovery after shear, as exemplified in the Examples section thatfollows.

Viscosity is a measure of the resistance of a fluid which is beingdeformed by, for example, shear stress. Shear viscosity measures thereaction to applied shear stress; and represents the ratio between thepressure exerted on the surface of a fluid, to the change in velocity ofthe fluid down the fluid. Shear viscosity is typically measured at anelevating shear rate, as exemplified in the Examples section thatfollows. Values are therefore indicated for a specific shear rate.

In some embodiments, the biocompatible polymer has a relatively lowshear viscosity, particularly compared to hydrogels.

In some embodiments, a biocompatible polymer, although characterized asviscoelastic, does not form a hydrogel in an aqueous solution.

In some embodiments, the biocompatible polymer is water-soluble.

Selecting biocompatible polymers suitable for forming the hydrogelhybrids described herein should be evident to any person skilled in theart in view of the guidelines provided herein.

Exemplary biocompatible polymers that are suitable for use in thecontext of embodiments of the present invention are polysaccharides.

The term “polysaccharide” as used herein is meant to include compoundscomposed of 10 saccharide units and up to hundreds and even thousands ofmonosaccharide units per molecule, which are held together by glycosidebonds and range in their molecular weights from around 5,000 and up tomillions of Daltons.

Polysaccharides that have desired physical properties, as defined hereinwith respect to swelling capability and viscoelasticity, are typicallyhighly hydrated polysaccharides, or highly hydrated linearpolysaccharides, as described hereinabove.

In some embodiments, the polysaccharide is a GAG. As noted hereinabove,GAGs are natural polymers which are major components of the nativeextracellular matrix (ECM), and are known to support enhanced cellattachment and proliferation.

In some embodiments, the polysaccharide is hyaluronic acid (HA).

In some embodiments, the polysaccharide is chitosan.

Additional suitable polysaccharides include, but are not limited to,agar, alginate, starch, laminarin and pectin, as long as these polymersexhibit the desired physical characteristics.

Additional exemplary suitable biocompatible polymers include, but arenot limited to, proteins such as collagen, elastin and fibrin.

The biocompatible polymers can be a natural polymer or a syntheticpolymer.

In some embodiments, the biocompatible polymer of choice has an averagemolecular weight that ranges from 1 kDa to 10,000 kDa. Without beingbound by any particular theory, it is suggested that the molecularweight of the polymer affects the physical properties of the formedhybrid hydrogel, and can be selected so to impart the desired propertiesto the hydrogel.

For example, it is suggested that using polymers with higher molecularweight results in hybrid hydrogels with higher viscosity and vice versa.

In some embodiments, high molecular weight polymers, e.g., having amolecular weight higher than 10 kDa, are used.

In some embodiments, the hybrid hydrogel comprises more than one type ofa biocompatible polymer as described herein.

Without being bound by any particular theory, the hybrid hydrogelsdescribed herein comprise fibrous networks which can be composed ofpeptide fibrils formed upon self-assembly of the peptides, with thebiocompatible polymer being encaged therewithin, or, alternatively or inaddition, can be composed of fibrous peptide structures having entangledtherewith the polymer.

According to some embodiments of the present invention, the fibrilscomposing the hydrogel have an average diameter or a cross-section ofless than 1 μm. In some embodiments, the fibrils have an averagediameter that ranges from about 1 nm to about 500 nm, more preferablyfrom about 10 nm to about 500 nm, more preferably from about 10 nm toabout 200 nm and more preferably from about 10 nm to about 100 nm.

As used herein, the phrase “a plurality of peptides capable ofself-assembling in an aqueous solution” encompasses any peptides thatunder certain conditions (e.g., concentration and/or temperature),spontaneously rearrange so as to form peptide fibrils that form thehydrogel's fibrous network.

The term “peptide” as used herein encompasses native peptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and peptidomimetics (typically, synthetically synthesizedpeptides), as well as peptoids and semipeptoids which are peptideanalogs, which may have, for example, modifications rendering thepeptides more stable while in a body or more capable of penetrating intocells. Such modifications include, but are not limited to, N-terminusmodification, C-terminus modification, peptide bond modification,including, but not limited to, CH₂—NH, CH—S, CH₂—S═O, O═C—NH, CH₂—O,CH₂—CH₂, S═C—NH, CH═CH or CF═CH, backbone modifications, and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified, for example, in Quantitative DrugDesign, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press(1992), which is incorporated by reference as if fully set forth herein.Further details in this respect are provided hereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH₃)—CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH₂—), a-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH₂—NH—), hydroxyethylene bonds (—CH(OH)—CH₂—), thioamide bonds(—CS—NH—), olefinic double bonds —CH=CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH₂—CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom. These modifications canoccur at any of the bonds along the peptide chain and even at several(2-3) at the same time.

As used herein throughout, the term “amino acid” or “amino acids” isunderstood to include the 20 naturally occurring amino acids; thoseamino acids often modified post-translationally in vivo, including, forexample, hydroxyproline, phosphoserine and phosphothreonine; and otherunusual amino acids including, but not limited to, 2-aminoadipic acid,hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.Furthermore, the term “amino acid” includes both D- and L-amino acids.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic unnatural acids such as phenylglycine, TIC, naphthylalanine(Nal), ring-methylated derivatives of Phe, halogenated derivatives ofPhe or 0-methyl-Tyr, and β amino-acids.

In addition to the above, the peptides may also include one or moremodified amino acids (e.g., biotinylated amino acids) or one or morenon-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).

As discussed hereinabove, it has been shown previously the relativelyshort aromatic peptides can self-assemble into hydrogels, presumably dueto aromatic interactions.

In some embodiments, the peptides used for forming the hybrid hydrogelsdescribed herein have at least two amino acid residues and up to 6 aminoacid residues, provided that at least one amino acid residue, in eachpeptide of the plurality of peptides used, is an aromatic amino acid.Thus, each of the peptides used for forming the hydrogels describedherein can have two, three, four, five or six amino acid residues.

The peptides used for forming the hydrogels described herein aretherefore relatively short peptides. Using such relatively shortpeptides is highly advantageous, allowing the formation of complexpeptide nanostructures and fibrous networks from relatively cheap andreadily available simple building blocks.

Each peptide in the plurality of peptides used for forming the hydrogelcomprises at least one aromatic amino acid residue

In some embodiments of the present invention, at least one peptide inthe plurality of peptides used for forming the hydrogel is apolyaromatic peptide, comprising two or more aromatic amino acidresidues. In some embodiments, at least one peptide in the plurality ofpeptides consists essentially of aromatic amino acid residues. In someembodiments, each peptide in the plurality of peptides consistsessentially of aromatic amino acid residues.

Thus, for example, the peptides used for forming the hybrid hydrogeldescribed herein can include any combination of: dipeptides composed ofone or two aromatic amino acid residues; tripeptides including one, twoor three aromatic amino acid residues; tetrapeptides including two,three or four aromatic amino acid residues; pentapeptides including two,three, four or five aromatic amino acid residues; and hexapeptidesincluding two, three, four, five or six aromatic amino acid residues.

In some embodiments, one or more peptides in the plurality of peptidesused for forming the hybrid hydrogel include two amino acid residues,and hence is a dipeptide.

In some embodiments, each of the peptides used for forming the hybridhydrogel comprises two amino acid residues and therefore the hybridhydrogel is formed from a plurality of dipeptides.

Each of these dipeptides can include one or two aromatic amino acidresidues. Preferably, each of these dipeptides includes two aromaticamino acid residues. The aromatic residues composing the dipeptide canbe the same, such that the dipeptide is a homodipeptide, or different.In some embodiments, the hydrogel is formed from homodipeptides.

Hence, in some embodiments of the present invention, each peptide in theplurality of peptides used for forming the hybrid hydrogel is ahomodipeptide composed of two aromatic amino acid residues that areidentical with respect to their side-chains residue.

The phrase “aromatic amino acid residue”, as used herein, refers to anamino acid residue that has an aromatic moiety in its side-chain.

As used herein, the phrase “aromatic moiety” describes a monocyclic orpolycyclic moiety having a completely conjugated pi-electron system. Thearomatic moiety can be an all-carbon moiety or can include one or moreheteroatoms such as, for example, nitrogen, sulfur or oxygen. Thearomatic moiety can be substituted or unsubstituted, whereby whensubstituted, the substituent can be, for example, one or more of alkyl,trihaloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, halo, nitro, azo, hydroxy, alkoxy, thiohydroxy,thioalkoxy, cyano and amine.

Exemplary aromatic moieties include, but are not limited to, phenyl,biphenyl, naphthalenyl, phenanthrenyl, anthracenyl,[1,10]phenanthrolinyl, indoles, thiophenes, thiazoles and,[2,2′]bipyridinyl, each being optionally substituted. Thus,representative examples of aromatic moieties that can serve as the sidechain within the aromatic amino acid residues described herein include,without limitation, substituted or unsubstituted naphthalenyl,substituted or unsubstituted phenanthrenyl, substituted or unsubstitutedanthracenyl, substituted or unsubstituted [1,10]phenanthrolinyl,substituted or unsubstituted [2,2′]Thipyridinyl, substituted orunsubstituted biphenyl and substituted or unsubstituted phenyl. Thearomatic moiety can alternatively be substituted or unsubstitutedheteroaryl such as, for example, indole, thiophene, imidazole, oxazole,thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline,quinazoline, quinoxaline, and purine.

As used herein, the term “alkyl” refers to a saturated aliphatichydrocarbon including straight chain and branched chain groups.Preferably, the alkyl group has 1 to 20 carbon atoms. The alkyl groupmay be substituted or unsubstituted. When substituted, the substituentgroup can be, for example, trihaloalkyl, alkenyl, alkynyl, cycloalkyl,aryl, heteroaryl, heteroalicyclic, halo, nitro, azo, hydroxy, alkoxy,thiohydroxy, thioalkoxy, cyano, and amine.

A “cycloalkyl” group refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereinone or more of the rings does not have a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. Acycloalkyl group may be substituted or unsubstituted. When substituted,the substituent group can be, for example, alkyl, trihaloalkyl, alkenyl,alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, nitro,azo, hydroxy, alkoxy, thiohydroxy, thioalkoxy, cyano, and amine.

An “alkenyl” group refers to an alkyl group which consists of at leasttwo carbon atoms and at least one carbon-carbon double bond.

An “alkynyl” group refers to an alkyl group which consists of at leasttwo carbon atoms and at least one carbon-carbon triple bond.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, naphthalenyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted, the substituent group can be, for example, alkyl,trihaloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, halo, nitro, azo, hydroxy, alkoxy, thiohydroxy,thioalkoxy, cyano, and amine.

A “heteroaryl” group refers to a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine. The heteroaryl group may besubstituted or unsubstituted. When substituted, the substituent groupcan be, for example, alkyl, trihaloalkyl, alkenyl, alkynyl, cycloalkyl,aryl, heteroaryl, heteroalicyclic, halo, nitro, azo, hydroxy, alkoxy,thiohydroxy, thioalkoxy, cyano, and amine.

A “heteroalicyclic” group refers to a monocyclic or fused ring grouphaving in the ring(s) one or more atoms such as nitrogen, oxygen andsulfur. The rings may also have one or more double bonds. However, therings do not have a completely conjugated pi-electron system. Theheteroalicyclic may be substituted or unsubstituted. When substituted,the substituted group can be, for example, lone pair electrons, alkyl,trihaloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, halo, nitro, azo, hydroxy, alkoxy, thiohydroxy,thioalkoxy, cyano, and amine. Representative examples are piperidine,piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the like.

A “hydroxy” group refers to an —OH group.

A “thio” group (also referred to herein, interchangeably as “thiol” or“thiohydroxy”) refers to a —SH group.

An “azide” group refers to a —N═N≡N group.

An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group,as defined herein.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,as defined herein.

A “thioalkoxy” group refers to both an —S-alkyl group, and an—S-cycloalkyl group, as defined herein.

A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroarylgroup, as defined herein.

A “halo” or “halide” group refers to fluorine, chlorine, bromine oriodine.

A “halophenyl” group refers to a phenyl substituted by two, three, fouror five halo groups, as defined herein.

A “trihaloalkyl” group refers to an alkyl substituted by three halogroups, as defined herein. A representative example is trihalomethyl. An“amino” group refers to an —NR′R″ group where R′ and R″ are hydrogen,alkyl, cycloalkyl or aryl.

A “nitro” group refers to an —NO₂ group.

A “cyano” group refers to a —C≡N group.

The hydrogels of the present invention can be composed of linear orcyclic peptides (e.g., cyclic di-peptides of phenylalanine).

According to some embodiments of the present invention, one or morepeptides in the plurality of peptides used to form the hydrogeldescribed herein is an end-capping modified peptide.

The phrase “end-capping modified peptide”, as used herein, refers to apeptide which has been modified at the N-(amine)terminus and/or at theC-(carboxyl)terminus thereof. The end-capping modification refers to theattachment of a chemical moiety to the terminus, so as to form a cap.Such a chemical moiety is referred to herein as an end-capping moietyand is typically also referred to herein and in the art,interchangeably, as a peptide protecting moiety or group.

The phrase “end-capping moiety”, as used herein, refers to a moiety thatwhen attached to the terminus of the peptide, modifies the end-capping.The end-capping modification typically results in masking the charge ofthe peptide terminus, and/or altering chemical features thereof, suchas, hydrophobicity, hydrophilicity, reactivity, solubility and the like.Examples of moieties suitable for peptide end-capping modification canbe found, for example, in Green et al., “Protective Groups in OrganicChemistry”, (Wiley, 2.sup.nd ed. 1991) and Harrison et al., “Compendiumof Synthetic Organic Methods”, Vols. 1-8 (John Wiley and Sons,1971-1996).

Representative examples of N-terminus end-capping moieties include, butare not limited to, formyl, acetyl (also denoted herein as “Ac”),trifluoroacetyl, benzyl, benzyloxycarbonyl (also denoted herein as“Cbz”), tert-butoxycarbonyl (also denoted herein as “Boc”),trimethylsilyl (also denoted “TMS”), 2-trimethylsilyl-ethanesulfonyl(also denoted “SES”), trityl and substituted trityl groups,allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (also denoted herein as“Fmoc”), and nitro-veratryloxycarbonyl (“NVOC”).

Representative examples of C-terminus end-capping moieties are typicallymoieties that lead to acylation of the carboxy group at the C-terminusand include, but are not limited to, benzyl and trityl ethers as well asalkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers, allylethers, monomethoxytrityl and dimethoxytrityl. Alternatively the —COOHgroup of the C-terminus end-capping may be modified to an amide group.

Other end-capping modifications of peptides include replacement of theamine and/or carboxyl with a different moiety, such as hydroxyl, thiol,halide, alkyl, aryl, alkoxy, aryloxy and the like, as these terms aredefined herein.

In a preferred embodiment of the present invention, all of the peptidesthat comprise the hydrogels are end-capping modified only at theN-termini

However, other combinations of N-terminus end capping and C-terminus endcapping of the various peptides composing the hydrogel are alsocontemplated. These include, for example, the presence of certainpercents of end-capping modified peptides within the plurality ofpeptides, whereby the peptides are modified at the N-termini and/or theC-termini.

Another chemical property of an end-capping of a peptide is itshydrophobic/hydrophilic nature, which when unmodified, is hydrophilic inpeptides. Altering the hydrophobic/hydrophilic property of one or bothof the end-capping of the peptide may result, for example, in alteringthe morphology of the resulting fibrous network.

End-capping moieties can be further classified by their aromaticity.Thus, end-capping moieties can be aromatic or non-aromatic.

Representative examples of non-aromatic end capping moieties suitablefor N-terminus modification include, without limitation, formyl, acetyltrifluoroacetyl, tert-butoxycarbonyl, trimethylsilyl, and2-trimethylsilyl-ethanesulfonyl. Representative examples of non-aromaticend capping moieties suitable for C-terminus modification include,without limitation, amides, allyloxycarbonyl, trialkylsilyl ethers andallyl ethers.

Representative examples of aromatic end capping moieties suitable forN-terminus modification include, without limitation,fluorenylmethyloxycarbonyl (Fmoc). Representative examples of aromaticend capping moieties suitable for C-terminus modification include,without limitation, benzyl, benzyloxycarbonyl (Cbz), trityl andsubstituted trityl groups.

In some embodiments of the present invention, the end-capping modifiedpeptides are modified by an aromatic (e.g. Fmoc) end-capping moiety. Itis assumed that such an aromatic end-capping moiety also participates inthe aromatic interactions, thus contributing to the formation andproperties of the hybrid hydrogel.

The end-capping modified peptides utilized according to the presentembodiments can be collectively represented by the following generalFormula I:

R₁—[A₁]-[A₂]— . . . [A_(n)]—R₂   Formula I

wherein:

n is an integer from 2 to 6;

A₁, A₂, . . . , A_(n) are each independently an amino acid residue asthis term is defined herein, providing that at least one of A₁, A₂, . .. , A_(n) is an aromatic amino acid residue as this term is definedherein;

R₁ is an N-terminus end-capping moiety or absent; and

R₂ is a C-terminus end-capping moiety or absent.

As described hereinabove, according to some embodiments of the presentinvention, the hydrogel comprises one or more end-capping modifiedhomodipeptide.

Representative examples of end-capping modified homodipeptides include,without limitation, an end-capping modifiednaphthylalanine-naphthylalanine dipeptide,phenanthrenylalanine-phenanthrenylalanine dipeptide,anthracenylalanine-anthracenylalanine dipeptide,[1,10]phenanthrolinylalanine-[1,10]phenanthrolinylalanine dipeptide,[2,2′]bipyridinylalanine-[2,2′]bipyridinylalanine dipeptide,(pentahalo-phenylalanine)-(pentahalo-phenylalanine) dipeptide,phenylalanine-phenylalanine dipeptide,(amino-phenylalanine)-(amino-phenylalanine) dipeptide,(dialkylamino-phenylalanine)-(dialkylamino-phenylalanine) dipeptide,(halophenylalanine)-(halophenylalanine) dipeptide,(alkoxy-phenylalanine)-(alkoxy-phenylalanine) dipeptide,(trihalomethyl-phenylalanine)-(trihalomethyl-phenylalanine) dipeptide,(4-phenyl-phenylalanine)-(4-phenyl-phenylalanine) dipeptide and(nitro-phenylalanine)-(nitro-phenylalanine) dipeptide, whereby thesehomodipeptides are preferably end-capping modified by an aromaticmoiety, and more preferably, are end-capping modified at the N-terminusthereof by an aromatic moiety such as Fmoc.

The end-capping modification of the peptides forming the hybrid hydrogeldescribed herein can be further utilized for incorporating into thehydrogel a labeling moiety, as is detailed hereinbelow. Thus, accordingto an embodiment of the present invention, the one or more end-cappingmodified peptide comprises a labeling moiety. The labeling moiety canform a part of the end-capping moiety or can be the end-capping moietyitself.

In some embodiments of the present invention, the plurality of peptidesin the hybrid hydrogel described herein comprises, or consistsessentially of, a plurality of dipeptides, each comprising at least onearomatic amino acid residue.

In some embodiments, in each of the dipeptides both amino acid residuesare aromatic amino acid residues, as described herein.

In some embodiments, in each of the dipeptides, the aromatic amino acidresidues are the same, such that each of the dipeptides is ahomodipeptide, or an aromatic homodipeptide.

In some embodiments, the plurality of dipeptides comprises, or consistsessentially of, a plurality of Phe-Phe homodipeptides.

In some embodiments, the plurality of dipeptides comprises, or consistsessentially of, a plurality of homodipeptides (e.g., Phe-Phe) which areend-capping modified peptides, wherein the end-capping moiety in suchpeptides in an aromatic moiety such as F-moc.

In some embodiments, the plurality of peptides comprises, or consistsessentially of, a plurality of Fmoc-Phe-Phe (Fmoc-FF).

In some embodiments, the hydrogel comprises a fibrous network of aplurality of dipeptides, as described herein (e.g., Fmoc-FF), andhyaluronic acid.

In some embodiments, the hydrogel comprises a fibrous network of aplurality of dipeptides, as described herein (e.g., Fmoc-FF), andchitosan.

In some embodiments, the hydrogel comprises a fibrous network of aplurality of dipeptides, as described herein (e.g., Fmoc-FF), and anyother biocompatible polymer, as described herein.

For each of hybrid hydrogels described herein, the total concentrationof the peptide content (the plurality of peptides) and the biocompatiblepolymer ranges from 0.1 weight percent to 5 weight percents of the totalweight of the gel, with the balance being water or an aqueous solution(e.g., a buffer).

Without being bound by any particular theory, it is suggested that athigher concentrations of the components forming the hydrogel, hydrogelwith enhanced mechanical strength are obtained. On the other hand, toohigh concentrations of the components forming the hydrogel may interferewith the intermolecular interactions for forming the gel. This is morerelevant to polymers with high molecular weight (such as HA). Thus, incase of polymers with molecular weight than is lower, for example, than100 kDa, higher concentrations of the components can be used.

In some embodiments, the total concentration of the components rangesfrom 0.5 weight percent to 2.5 weight percent of the total weight of thegel.

In embodiments where the biocompatible polymer is hyaluronic acid, thetotal concentration of the peptide content (the plurality of peptides)and the biocompatible polymer ranges from 0.5 to 1 weight percent of thetotal weight of the gel.

In view of the surprising findings that the biological and physicalproperties of the hybrid hydrogels described herein are averagable, theratio between the components in the gel also determines the propertiesof the obtained hybrid hydrogel and can be manipulated so as to controlthe physical and biological properties of the hybrid hydrogel.

In general, hybrid hydrogels with higher peptide content arecharacterized by higher storage shear modulus (G′), higher viscosity,lower elasticity and lower swelling ratio. Hybrid hydrogels with highercontent of the biocompatible polymer are characterized by lower storageshear modulus (G′), lower viscosity, higher elasticity and higherswelling ratio.

The weight ratio of the dipeptides and the biocompatible polymer canrange from 20:1 to 1:20, from 10:1 to 1:10, from 8:1 to 1:8, from 5:1 to1:5, from 4:1 to 1:4, from 3:1 to 1:3, from 2:1 to 1:2 and can be 1:1.

In some embodiments, the weight ratio of the dipeptides and thebiocompatible polymer is 1:1. In such hybrid hydrogels, the biologicaland physical properties are averaged between the components.

In some embodiments, the weight ratio of the dipeptides and thebiocompatible polymer ranges from 3:1 to 1:3.

In exemplary embodiments, the total weight of the peptides and thepolymer is 0.5 weight percent and the ratio between components rangesfrom 3:1 to 1:3.

As noted hereinabove, the hybrid hydrogels described herein can beregarded as means to enhance mechanical properties of biocompatiblepolymers, at one hand, and to improve biocompatibility and malleabilityof the peptide hydrogels, on the other hand.

Thus, in some embodiments, the hybrid hydrogel as described herein ischaracterized by a storage modulus G′ to loss modulus G″ ratio that ishigher by at least 2-folds than such a ratio of a corresponding aqueoussolution of the biocompatible polymer. By “corresponding aqueoussolution” it is meant a solution with a concentration of the polymer orthe peptides that is similar or the same as the total concentration ofthe polymer or the peptides in the hybrid hydrogel.

In many inert and living materials, the relationship between elastic andfrictional stresses turns out to be very nearly invariant. The ratiobetween the elastic (storage) and frictional (loss) moduli is called thehysteresivity, h, or, equivalently, the structural damping coefficient.Thus, for each unit of peak elastic strain energy that is stored duringa cyclic deformation, 10 to 20 percents of that elastic energy is taxedas friction and lost irreversibly to heat.

In systems conforming to the structural damping law, the hysteresivity his constant with or insensitive to changes in oscillatory frequency, andthe loss modulus G″ becomes a constant fraction of the elastic modulus.

The hysteresivity represents the fraction of the elastic energy that islost to heat, and is an intensive property that is dimensionless.

Higher storage to loss modulus ratio therefore indicates the formationof stronger and more rigid hydrogel.

In some embodiments, the hybrid hydrogel described herein ischaracterized by a shear storage modulus (G′) that is lower by at least10% of the storage modulus G′ of a hydrogel formed from a correspondingaqueous solution of the plurality of peptides.

In some embodiments, the storage modulus of the hybrid hydrogel is lowerby 10%, 20%, 30%, 40% and even 50% percents lower than the storagemodulus of a hydrogel formed from a corresponding aqueous solution ofthe plurality of peptides.

Thus, the less rigid structure of the hybrid hydrogel compared to acorresponding hydrogel made from peptides without the polymer,advantageously renders the hybrid hydrogel malleable.

In some embodiments, the hybrid hydrogel described herein ischaracterized by a storage modulus G′ that is higher by at least 5-foldsof a storage modulus G′ of a corresponding aqueous solution of thebiocompatible polymer.

In some embodiments, the hybrid hydrogel is characterized by a storagemodulus G′ that is a 5-folds, 10-folds, 20-folds, 30-folds, 50-folds,60-folds, 60-folds and even 100-folds higher than that of acorresponding aqueous solution of the biocompatible polymer.

The improved malleability of the hybrid hydrogels described herein isfurther demonstrated by its improved viscoelastic properties compared toeach of the components when used alone in an aqueous solution.

In some embodiments, the hybrid hydrogel is characterized by a viscosityhigher by at least 10% of a viscosity of the biocompatible polymer, whenmeasured at an indicated shear rate.

Depending on the shear rate for a measured viscosity, the viscosity ofthe hybrid hydrogel can be higher by 10%, by 20%, by 50%, by 80%, or by100% of that of a corresponding aqueous solution of the biocompatiblepolymer, and can be even 3-folds, 4-folds, 5-folds, 10-folds and even100-folds or 1000-folds higher.

In some embodiments, the hybrid hydrogel is characterized by a viscositychange through time higher by at least 2-folds than a viscosity changethrough time of the biocompatible polymer, indicating less elasticity ofthe hybrid hydrogel compared to the biocompatible polymer.

In some embodiments, the hybrid hydrogel is characterized by viscosityrecovery after shear that is at least 2-folds, or at least 3-folds, orat least 5-folds, or at least 10-folds, and can also be as high as100-folds higher than that of a hydrogel formed from a correspondingaqueous solution of the peptides, indicating higher elasticity of hybridhydrogel compared to a peptide hydrogel. Also here, the differences ofviscosity recovery after shear are as determined for a viscositymeasured certain shear rate.

In some embodiments, the hybrid hydrogel is characterized by a swellingratio (Q) higher by at least 5%, or at least 10%, or at least 20%, or atleast 50% of a swelling ratio of a hydrogel formed of a correspondingaqueous solution of the plurality of peptides. Higher swellingcapability renders materials more suitable for biomaterial applications,as discussed hereinabove.

Indeed, in some embodiments, a hybrid hydrogel as described herein ischaracterized by biocompatibility to cell viability higher by at least2-folds than a biocompatibility to cell viability of a hydrogel formedof the plurality of peptides.

By “biocompatibility to cell viability” it is meant the percents ofcells that remain viable at an indicated time point post contacting thecells with the hydrogel, and is representing the suitability of thehybrid hydrogel as a matrix for cell growth.

In some embodiments, the hybrid hydrogel described herein ischaracterized as maintaining cells therewithin to an extent that ishigher by at least 5%, at least 10%, at least 20%, at least 30% and evenby at least 50% than that of a corresponding aqueous solution of thebiocompatible polymer, indicating an improved capability of the hybridhydrogel to serve as a scaffold, as defined herein, compared to thebiocompatible polymer.

Further, as discussed hereinabove in the context of HA, biocompatiblepolymers are often biodegradable. Natural polymers are often furthercharacterized by relatively fast biodegradation due to enzymaticdegradation thereof. The fast biodegradation impediment the performanceof such polymers in various applications.

As exemplified herein, biodegradation of such polymers is substantiallyreduced when combined into the hybrid hydrogels described herein.

Hence, in some embodiments, the hybrid hydrogels described herein arecharacterized by a biodegradation rate that is lower by at least2-folds, at least 3-folds, at least 4-folds, at least 5-folds, at least8-folds and even at least 10-folds or higher fold lower than thebiodegradation rate of the biocompatible polymer.

Biodegradation rate can be determined by the time 50% of mass loss of asubstance is observed; or, for example, by the degree of mass loss(e.g., as percentage of original mass) upon incubation for 7 days in aphysiological medium.

By controllably averaging the biological and physical properties of itscomponents, hybrid hydrogels as described herein feature such propertiesthat are highly advantageous for biomaterial applications.

In some embodiments, a hybrid hydrogel as described herein ischaracterized by a storage modulus G′ to loss modulus G″ ratio that isgreater than 1, greater than 2, greater than 3, greater than 4, and evengreater than 5.

In some embodiments, a hybrid hydrogel as described herein ischaracterized by a storage modulus G′ higher than 1,000 Pa at 10 Hzfrequency and at 25° C.

In some embodiments, a hybrid hydrogel as described herein ischaracterized by a storage modulus G′ lower than 100,000 Pa at 10 Hzfrequency and at 25° C.

In some embodiments, a hybrid hydrogel as described herein ischaracterized by a viscosity that ranges from 200 to 2000 Pa·s at 0.1Sec⁻¹ shear rate, at 25 ° C.

In some embodiments, a hybrid hydrogel as described herein ischaracterized by a viscosity change through time, as defined herein,higher than 1%, higher than 2%, and can be higher than 3% and evenhigher.

In some embodiments, a hybrid hydrogel as described herein ischaracterized by a viscosity recovery after shear that is at least 20%,at least 30%, at least 40% or at least 50% after 10 minutes at 0.1sec⁻¹.

In some embodiments, a hybrid hydrogel as described herein ischaracterized by a swelling ratio (Q) that ranges from 100 to 500.

Notably, in some embodiments, the hybrid hydrogels described herein aredevoid of a chemical cross-linking agent. Without being bound by anyparticular theory, it is suggested that the polymer and peptides arelinked to one another in the hybrid hydrogel by a physical crosslinking.

It should be noted that the structural, physical and chemical propertiesof the hybrid hydrogels described herein can be controlled andmanipulated by employing different peptide building blocks, by alteringthe types of functional groups therein, and by varying the type ofend-capping moiety used, and/or by employing different biocompatiblepolymers, and/or by altering their molecular weight, as well as bymanipulating various parameters in their preparation (e.g.,concentration of each component or total concentration of bothcomponents).

As demonstrated in the Examples section that follows, the hybridhydrogels presented herein are formed in an aqueous solution thatcomprises the plurality of peptides and the biocompatible polymer.

Thus, according to another aspect of the present invention there isprovided a process of preparing the hybrid hydrogels described herein.In some embodiments, the process is effected by contacting a pluralityof peptides and the biocompatible polymer, as described in detailhereinabove, with an aqueous solution.

In some embodiments, the process is effected by dissolving the polymerin the aqueous solution and contacting the peptides with the aqueoussolution in which the polymer is dissolved.

In some embodiments, the plurality of peptides is dissolved in awater-miscible solvent, prior to contacting the plurality of peptideswith the polymer and the aqueous solution.

Contacting a solution of the peptides dissolved in an organic solventwith an aqueous solution comprising dissolved polymer can be regarded asdiluting the peptide's solution to a concentration that allowsself-assembly of the peptides.

The phrase “water-miscible organic solvent”, as used herein, refers toorganic solvents that are soluble in water. Several factors inherent inthe structure of the solvent molecules can affect the miscibility oforganic solvents in water, such as for example, the length of the carbonchain and the type of functional groups therein. Hydrogen bonding playsa key role in making organic solvents miscible in water. For example, inalcohols, the hydroxyl group can form hydrogen bonding with watermolecules. In addition, aldehydes, ketones and carboxylic acids can formhydrogen bonding via the carbonyl oxygen. Hydrogen bonding between etherand water molecules is also possible, enabling some degree ofmiscibility of simple ethers in water.

Examples of water-miscible organic solvents include, without limitation,simple alcohols, such as, methanol, ethanol, 1-propanol, 2-propanol,1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol,2,2-dimethyl-1-propanol and their halogen substituted analogues,ethylene glycol, acetone, dimethylsulfoxide, acetic acid diethyl ether,tetrahydrofuran etc.

Representative examples of organic solvents that were successfullypracticed in generating exemplary hydrogels according to the presentinvention include, acetone, dimethylsulfoxide and hexafluoroisopropanol(e.g., 1,1,1,3,3,3-hexafluoro-2-propanol, abbreviated herein as HFIP).

In some embodiments, the organic solvent is dimethylsulfoxide(abbreviated DMSO).

As discussed hereinabove, the hydrogels' properties can be manipulatedby controlling the concentration of each of the components; the peptidesand the biocompatible polymer, individually and as a total concentrationin the solution.

In some embodiments, the total concentration of the plurality ofpeptides and the polymer and in the aqueous solution ranges from about0.1 mg/ml to about 50 mg/ml, or from 0.1 mg/ml to 25 mg/ml, or from 0.1mg/ml to 10 mg/ml.

The final concentration of the plurality of peptide and of thebiocompatible polymer in the aqueous solution and hence in the generatedhybrid hydrogel can be readily determined by determining theconcentration of each component in the aqueous solution.

For example, one can prepare a stock solution of the peptides in awater-miscible organic solvent or in a solvent mixture of such anorganic solvent and water, in a certain concentration of the peptides,and a stock aqueous solution in which the polymer is dissolved in thesame concentration of the polymer as for the peptide solution, and thenmix the desired relative amounts of the solutions, so as to determinethe ratio between the components in hybrid hydrogel. The concentrationof each component in the solution will determine the final totalconcentration of the components in the hybrid hydrogel. Othermanipulations of the concentration of each component in its stocksolution and of the ratio between the stock solutions when contacted arealso contemplated.

In some embodiments, the aqueous solution is a buffer (e.g., PBS).

The process of generating the hybrid hydrogels described hereinabove ispreferably performed at room temperature. Alternatively, it can beeffected at a physiological temperature (e.g., at 37° C.).

In some embodiments, contacting is further effected by mixing the formedaqueous solution (containing both components). Mixing can be performed,for example, by manual or mechanical shaking (e.g., by vortex), or bymagnetic or mechanical stirring. In some embodiments, missing isperformed by means of vortex.

Notably, the hybrid hydrogels described herein are generated withoutusing a chemical crosslinking agent.

Once both the peptides and the polymer are contacted with an aqueoussolution, and optionally the solution is mixed, a hydrogel is formed. Insome embodiments, a hydrogel is formed upon maintaining the mixture atroom temperature, for a time period that ranges from 1 minute to about 1hour.

According to some embodiments of the present invention, the preparationof the hydrogel is effected prior to its application to a desiredapplication site. Thus, for example, when the desired application siteis a bodily organ or cavity, the hydrogel is prepared ex-vivo, prior toits application, by contacting the plurality of peptides, the polymerand an aqueous solution, as described hereinabove, and is administeredsubsequent to its formation.

Alternatively, the preparation of the hybrid hydrogel can also beperformed upon its application, such that the plurality of peptides, thepolymer and the aqueous solution are each applied separately to thedesired site and the hybrid hydrogel is formed upon contacting thepeptides, the polymer and the aqueous solution at the desired site ofapplication. Thus, for example, contacting the peptides and the aqueoussolution can be performed in vivo, such that the plurality of peptides,the polymer and the aqueous solution are separately administered.

In some embodiments, in vivo contacting is effected by contacting thepeptides with an aqueous solution that comprises the polymer at thedesired site of application.

According to these embodiments, the administration is preferablyeffected locally, into a defined bodily cavity or organ, where theplurality of peptides, the polymer and the aqueous solution become incontact while maintaining the desired ratio therebetween that wouldallow the formation of a hydrogel within the organ or cavity. Asdiscussed hereinabove, the plurality of peptides can be utilized eitherper se, or, optionally and preferably, be dissolved in a water-miscibleorganic solvent, or any other suitable organic solvent, as describedhereinabove.

Using such a route of preparing the hybrid hydrogel in vivo allows tobeneficially utilize the formed hydrogel in biomaterial applicationssuch as, for example, dental procedures, as a dental implant or fillingmaterial, cosmetic or cosmeceutical applications, tissue regeneration,implantation, and in would healing, as a wound dressing that is formedat a bleeding site, as is further detailed hereinbelow.

The formation of the hydrogel can similarly be effected at other sitesof actions, other than a bodily organ or cavity, in which the hydrogelcan be beneficially utilized, according to the desired application. Suchapplications include, for example, nanoelectro- ormicroelecto-mechanical systems (also known as NEMS or MEMS,respectively).

Thus, according to another aspect of some embodiments of the presentinvention there is provided a kit for forming the hybrid hydrogeldescribed herein. In some embodiments, such a kit comprises a pluralityof peptides, as described herein, and a biocompatible polymer, asdescribed herein. In some embodiments, the kit further comprises anaqueous solution. In some embodiments, the peptides and the polymer areindividually packaged within the kit. When the kit further comprises anaqueous solution, each of the aqueous solution, the polymer and thepeptides can be individually packaged within the kit. Optionally, thepeptides and the aqueous solution are individually packaged within thekit, and the polymer is dissolved in the aqueous solution. In someembodiments, the peptides are dissolved in a water-miscible organicsolvent as described herein. In some embodiments, the peptide and theorganic solvent are individually packaged within the kit.

In some embodiments, the plurality of peptides, the polymer and theoptional aqueous solution and organic solvent are present within the kitin amounts that would allow generation of a hydrogel upon contactingthereof.

In some embodiments, the kit further comprises instructions forgenerating a desired hybrid hydrogel, either ex vivo or in vivo, asdescribed herein. The kit may further comprise instructions, andoptionally directives in the form of a table, how to use the componentsthereto to achieve a hybrid hydrogel with desired total concentration ofits components and/or with desired ratio of the peptides and thepolymer.

Such a kit can be utilized to prepare the hydrogel described herein atany of the desired site of actions (e.g., a bodily cavity or organ)described hereinabove.

The plurality of peptides in such a kit can be in a lyophilized form.

As used herein, the phrases “desired site of application” and “desiredapplication site” describe a site in which application of the hybridhydrogel described herein is beneficial, namely, in which the hydrogelcan be beneficially utilized for therapeutic, diagnostic, cosmetic,cosmeceutical and/or mechanical applications, as described in detailhereinbelow.

Such a kit can further comprise an active agent, as is detailedhereinbelow, which is incorporated in or on the hydrogel, upon itsformation, so as to form the composition-of-matter described herein.

The active agent can be individually packaged within the kit or can bepackaged along with the plurality of peptides, or along with the polymeror along with the aqueous solution.

As is further demonstrated in the Examples section that follows, thehydrogels formed according to the present invention are characterized byexceptional material properties, which render them highly advantageousfor use in applicative technologies.

Thus, for example, the hydrogels described herein are characterized bymalleability, which allows utilizing both their relative rigidity andelasticity. In some embodiments, the hybrid hydrogels are injectable andtherefore may be suitable for use in various medical and/or cosmeticapplications, as further discussed hereinbelow.

In some embodiments, the hybrid hydrogels described herein can beutilized as a matrix for encapsulating therein or attaching theretovarious agents. Indeed, it was shown that various substances can beembedded on and/or in the hydrogels. As demonstrated in the Examplessection that follows, the hybrid hydrogels enable to entrap thereinbiological substances such as cells, allowing expansion and elongationof the cells within the hydrogel.

Hence, according to another aspect of the present invention there isprovided a composition-of-matter, which comprises the hydrogel describedherein and at least one agent being incorporated therein and/or thereon.

As used herein, the term “incorporated” encompasses attachment,encapsulation, embedding, and entanglement and like interactions of theactive agent and the hybrid hydrogel, which can occur on a surface ofthe hydrogel, including outer surface or internal surface of the fibrousnetwork.

Agents that can be beneficially embedded in or on, or attached to, thehydrogel include, for example, therapeutically active agents, diagnosticagents, biological substances and labeling moieties. More particularexamples include, but are not limited to, drugs, cells, proteins,enzymes, hormones, growth factors, nucleic acids, organisms such asbacteria, fluorescence compounds or moieties, phosphorescence compoundsor moieties, and radioactive compounds or moieties.

As used herein, the phrase “therapeutically active agent” describes achemical substance, which exhibits a therapeutic activity whenadministered to a subject. These include, as non-limiting examples,inhibitors, ligands (e.g., receptor agonists or antagonists),co-factors, anti-inflammatory drugs (steroidal and non-steroidal),anti-psychotic agents, analgesics, anti-thrombogenic agents,anti-platelet agents, anti-coagulants, anti-diabetics, statins, toxins,antimicrobial agents, anti-histamines, metabolites, anti-metabolicagents, vasoactive agents, vasodilator agents, cardiovascular agents,chemotherapeutic agents, antioxidants, phospholipids, anti-proliferativeagents and heparins.

As used herein, the phrase “biological substance” refers to a substancethat is present in or is derived from a living organism or cell tissue.This phrase also encompasses the organisms, cells and tissues.Representative examples therefore include, without limitation, cells,amino acids, peptides, proteins, oligonucleotides, nucleic acids, genes,hormones, growth factors, enzymes, co-factors, antisenses, antibodies,antigens, vitamins, immunoglobulins, cytokines, prostaglandins,vitamins, toxins and the like, as well as organisms such as bacteria,viruses, fungi and the like.

As used herein, the phrase “diagnostic agent” describes an agent thatupon administration exhibits a measurable feature that corresponds to acertain medical condition. These include, for example, labelingcompounds or moieties, as is detailed hereinunder.

As used herein, the phrase “labeling compound or moiety” describes adetectable moiety or a probe which can be identified and traced by adetector using known techniques such as spectral measurements (e.g.,fluorescence, phosphorescence), electron microscopy, X-ray diffractionand imaging, positron emission tomography (PET), single photon emissioncomputed tomography (SPECT), magnetic resonance imaging (MRI), computedtomography (CT) and the like.

Representative examples of labeling compounds or moieties include,without limitation, chromophores, fluorescent compounds or moieties,phosphorescent compounds or moieties, contrast agents, radioactiveagents, magnetic compounds or moieties (e.g., diamagnetic, paramagneticand ferromagnetic materials), and heavy metal clusters, as is furtherdetailed hereinbelow, as well as any other known detectable moieties.

As used herein, the term “chromophore” refers to a chemical moiety orcompound that when attached to a substance renders the latter coloredand thus visible when various spectrophotometric measurements areapplied.

A heavy metal cluster can be, for example, a cluster of gold atoms used,for example, for labeling in electron microscopy or X-ray imagingtechniques.

As used herein, the phrase “fluorescent compound or moiety” refers to acompound or moiety that emits light at a specific wavelength duringexposure to radiation from an external source.

As used herein, the phrase “phosphorescent compound or moiety” refers toa compound or moiety that emits light without appreciable heat orexternal excitation, as occurs for example during the slow oxidation ofphosphorous.

As used herein, the phrase “radioactive compound or moiety” encompassesany chemical compound or moiety that includes one or more radioactiveisotopes. A radioactive isotope is an element which emits radiation.Examples include α-radiation emitters, β-radiation emitters orγ-radiation emitters.

While a labeling moiety can be attached to the hydrogel, in cases wherethe one or more of the peptides composing the hydrogel is an end-cappingmodified peptide, the end-capping moiety can serve as a labeling moietyper se.

Thus, for example, in cases where the Fmoc group described hereinaboveis used as the end-capping moiety, the end-capping moiety itself is afluorescent labeling moiety.

In another example, wherein the Fmoc described hereinabove furtherincludes a radioactive fluoro atom (e.g., ¹⁸F) is used as theend-capping moiety, the end-capping moiety itself is a radioactivelabeling moiety.

Other materials which may be incorporated in or on the hybrid hydrogeldescribed herein include, without limitation, conducting materials,semiconducting materials, thermoelectric materials, magnetic materials,light-emitting materials, biominerals, polymers and organic materials.

Each of the agents described herein can be incorporated in or on thehydrogel by means of chemical and/or physical interactions. Thus, forexample, compounds or moieties can be attached to the external and/orinternal surface of the hydrogel, by interacting with functional groupspresent within the hydrogel via, e.g., covalent bonds, electrostaticinteractions, hydrogen bonding, van der Waals interactions,donor-acceptor interactions, aromatic (e.g., π-π interactions, cation-πinteractions and metal-ligand interactions. These interactions lead tothe chemical attachment of the material to the fibrous network of thehybrid hydrogel.

As an example, various agents can be attached to the hydrogel viachemical interactions with the side chains, N-terminus or C-terminus ofthe peptides composing the hydrogel and/or with the end-cappingmoieties, if present.

Alternatively, various agents can be attached to the hydrogel byphysical interactions such as magnetic interactions, surface adsorption,encapsulation, entrapment, entanglement and the like.

Attachment of the various agents to the hybrid hydrogel can be effectedeither prior to or subsequent to the hydrogel formation. Thus, forexample, an agent or moiety can be attached to one or more of thepeptides composing the hydrogel prior to the hydrogel formation,resulting in a hydrogel having the agent attached thereto.Alternatively, an agent or moiety can be attached to surface groups ofthe hydrogel upon its formation.

Incorporation of the various agents can be effected by forming thehybrid hydrogel in a solution containing the incorporated agent.

Hydrogels entrapping therein a biological or chemical agent can bebeneficially utilized for encapsulation and controlled release of theagent.

Hydrogels having a labeling moiety attached thereto or encapsulatedtherein can be utilized in a variety of applications, including, forexample, tracing and tracking the location of the fibrous networks ofthe present invention in mechanical devices and electronic circuitry;and tracing, tracking and diagnosing concentrations of the hybridhydrogels of the present invention in a living tissue, cell or host.

As is further detailed in the Examples section that follows, it has beenshown that the hydrogel described herein can be utilized as a highlyefficient cell culture matrix, which maintains the cells viability,morphology and proliferation rate.

Hence, by being characterized by controllable, averagable biological andphysical properties, the hybrid hydrogels and composition-of-mattersdescribed herein can be beneficially utilized in various applications,as is detailed hereinunder.

The hybrid hydrogels or composition-of-matters described herein can, forexample, form a part of a pharmaceutical, cosmetic or cosmeceuticalcompositions, either alone or in the presence of a pharmaceutically orcosmetically acceptable carrier.

As used herein, a “pharmaceutical, cosmetic or cosmeceuticalcomposition” refers to a preparation of the hydrogel or thecomposition-of-matter described herein, with other chemical componentssuch as acceptable and suitable carriers and excipients. The purpose ofa pharmaceutical composition is to facilitate administration of acompound to an organism. The purpose of a cosmetic or cosmeceuticalcomposition is typically to facilitate the topical application of acompound to an organism, while often further providing the preparationwith aesthetical properties.

Hereinafter, the term “pharmaceutically, cosmetically or cosmeceuticallyacceptable carrier” refers to a carrier or a diluent that does not causesignificant irritation to an organism and does not abrogate thebiological activity and properties of the applied compound. Examples,without limitations, of carriers include propylene glycol, saline,emulsions and mixtures of organic solvents with water, as well as solid(e.g., powdered) and gaseous carriers.

The compositions described herein may be formulated in conventionalmanner using one or more acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the hydrogel intopreparations. Proper formulation is dependent upon the route ofadministration chosen.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition.

The pharmaceutical compositions described herein can be formulated forvarious routes of administration. Suitable routes of administration may,for example, include oral, sublingual, inhalation, rectal, transmucosal,transdermal, intracavemosal, topical, intestinal or parenteral delivery,including intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Formulations for topical administration include but are not limited tolotions, ointments, gels, creams, suppositories, drops, liquids, spraysand powders. Conventional carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, sachets,capsules or tablets. Thickeners, diluents, flavorings, dispersing aids,emulsifiers or binders may be desirable.

Formulations for parenteral administration may include, but are notlimited to, sterile solutions which may also contain buffers, diluentsand other suitable additives. Slow release compositions are envisagedfor treatment.

The compositions may, if desired, be presented in a pack or dispenserdevice, such as an FDA (the U.S. Food and Drug Administration) approvedkit, which may contain the hydrogel. The pack may, for example, comprisemetal or plastic foil, such as, but not limited to a blister pack or apressurized container (for inhalation). The pack or dispenser device maybe accompanied by instructions for administration. The pack or dispensermay also be accompanied by a notice associated with the container in aform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals, which notice is reflective of approval bythe agency of the form of the compositions for human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert.

Additionally, in some embodiments, the hybrid hydrogels,composition-of-matters or compositions described herein can be utilizedfor forming an article-of-manufacture, whereby thearticle-of-manufacture can be, for example, a cell culture matrix, aprotein microarray chip, a biosensor, a medicament, a drug deliverysystem, a cosmetic or cosmeceutical agent, an implant, an artificialbody part, a tissue engineering and regeneration system, and a wounddressing, as well as various medical devices.

Herein, the phase “cell culture matrix” refers to biocompatible naturaland synthetic matrix that can be used to create definedthree-dimensional (3D) microenvironment which allows cell growth. Thematrix optimally mimics the natural environment of the cells. Cellculture matrices are often used in tissue engineering.

As used herein, the phrase “protein microarray chip” refers to a solidbase, e.g., pieces of glass, on which different molecules of proteinhave been affixed at separate locations in an ordered manner, thusforming a microscopic array. In general, microarray chips aremeasurement devices used in biomedical applications to determine thepresence and/or amount of proteins in biological samples. Otherapplications include, for example, the identification of protein-proteininteractions, of substrates of protein kinases, or of targets ofbiologically active small molecules. Another use is as a base forantibodies, where the antibodies are spotted onto the protein chip andused as capture molecules to detect proteins from cell lysate solutions.As will be familiar to one ordinarily skilled in the art, the formationof high-density protein chips to fully understand protein function hadpreviously been a tremendous challenge. This is because proteins need tobe in a wet environment in order to remain structurally intact and carryout their biological functions. Since hydrogels allow the proteins toremain in a wet environment as described hereinabove, it is highlyadvantageous to use hydrogels in forming protein microarray chips.

Herein the term “biosensor” refers to a device that combines abiological component with a physicochemical detector component and whichis utilized for the detection of an analyte.

As used herein, the term “medicament” refers to a licensed drug taken tocure or reduce symptoms of an illness or medical condition.

As used herein, the phrase “drug delivery system” refers to a system fortransportation of a substance or drug to a specific location, and morespecifically, to a desired bodily target, whereby the target can be, forexample, an organ, a tissue, a cell, or a cellular compartment such asthe nucleus, the mitochondria, the cytoplasm, etc. This phrase alsorefers to a system for a controlled release of a substance or drug at adesired rate.

As used herein, the term “implant” refers to artificial devices ortissues which are made to replace and act as missing biologicalstructures. These include, for example, dental implants, artificial bodyparts such as artificial blood vessels or nerve tissues, bone implants,and the like.

As used herein, the phrase “tissue engineering and regeneration” refersto the engineering and regeneration of new living tissues in vitro,which are widely used to replace diseased, traumatized or otherunhealthy or unaesthetic tissues, collectively referred to herein as“damaged tissue”.

As used herein, the phrase “cosmetic or cosmeceutical agent” refers totopical substances that are utilized for aesthetical purposes.Cosmeceutical agents typically include substances that further exhibittherapeutic activity so as to provide the desired aesthetical effect.Cosmetic or cosmeceutical agents in which the hydrogels,compositions-of-matter and compositions described herein can bebeneficially utilized include, for example, agents for firming adefected skin or nail, make ups, gels, lacquers, eye shadows, lipglosses, lipsticks, and the like.

Medical devices in which the hydrogels, compositions-of-matter andcompositions described herein can be beneficially utilized include, forexample, anastomotic devices (e.g., stents), sleeves, films, adhesives,scaffolds and coatings.

Stents comprising the hydrogels, compositions-of-matter or compositionsdescribed herein can be used, for example, as scaffolds for intraluminalend to end anastomoses; as gastrointestinal anastomoses; in vascularsurgery; in transplantations (heart, kidneys, pancreas, lungs); inpulmonary airways (trachea, lungs etc.); in laser bonding (replacingsutures, clips and glues) and as supporting stents for keeping bodyorifices open.

Sleeves comprising the hydrogels, compositions-of-matter or compositionsdescribed herein can be used, for example, as outside scaffolds fornerves and tendon anastomoses.

Films comprising the hydrogels, compositions-of-matter or compositionsdescribed herein can be used, for example, as wound dressing, substratesfor cell culturing and as abdominal wall surgical reinforcement.

Coatings of medical devices comprising the hydrogels,compositions-of-matter or compositions described herein can be used torender the device biocompatible, having a therapeutic activity, adiagnostic activity, and the like.

Other devices include, for example, catheters, aortic aneurysm graftdevices, a heart valve, indwelling arterial catheters, indwelling venouscatheters, needles, threads, tubes, vascular clips, vascular sheaths anddrug delivery ports.

Other potential non pharmaceutical applications of the hydrogel of thepresent invention are related to the exceptional material properties ofthe hydrogel. These applications include, for example, employing thehydrogel in a vibration-damping device or in a packaging material.

As used herein, the term “vibration-damping device” refers to a devicewhich tends to reduce the amplitude of oscillations. Applicationsinclude for example the reduction of electric-signal (and hence sound)distortion in audio-electrical devices.

As used herein, the term “packaging material” refers to materialdesignated for the enclosing of a physical object, typically a productwhich needs physical protection.

Due to the high biocompatibility of the hybrid hydrogels describedherein, in particular cell viability-related biocompatibility, thesehybrid hydrogels, or scaffolds made therefrom, can be used for inducingtissue formation, either in vivo or ex vivo.

Thus, in some embodiments, the hybrid hydrogels described herein,compositions-of-matter, compositions, kits or articles containing thesame, can be used for inducing tissue formation or in the manufacture ofa medicament or an agent for inducing tissue formation, either in vivoor ex vivo.

The phrase “in vivo” refers to within a living organism such as a plantor an animal, preferably in mammals, preferably, in human subjects.

In vivo induction of tissue formation can be made by administering thehydrogel hybrid or a composition-of-matter comprising the hybridhydrogel, as described herein, to a desired sire of application, asdefined herein.

In some embodiments, administering can be effected by injection or byother implanting (e.g., by using a scalpel, spoon, spatula, or othersurgical device) the hydrogel at the desired site of application.

As used herein, the term “subject” refers to a vertebrate, preferably amammal, more preferably a human being (male or female) at any age.

Optionally, in vivo induction of tissue formation can be made byadministering to a subject the biocompatible hydrogel, the peptides andoptionally an aqueous solution, as described hereinabove forregenerating the hybrid hydrogel at a desired site of application.

In some embodiments, in vivo induction of tissue formation can beeffected by utilizing a composition-of-matter as described herein, whichcomprises the hybrid hydrogel as described herein and an active agentthat is useful in promoting or inducing tissue formation. Exemplary suchagents include, but are not limited to, growth factors, for example,insulin-like growth factor-1 (IGF-1), a transforming growth factor-β(TGF-β), a basic fibroblast growth factor (bFGF), a bone morphogenicprotein (BMP), a cartilage-inducing factor-A, a cartilage-inducingfactor-B, an osteoid-inducing factor, a collagen growth factor andosteogenin; and cells capable of promoting tissue formation, such as,but not limited to, stem cells such as embryonic stem cells, bone marrowstem cells, cord blood cells, mesenchymal stem cells, adult tissue stemcells, or differentiated cells such as neural cells, retina cells,epidermal cells, hepatocytes, fibroblasts, chondrocytes, pancreaticcells, osseous cells, cartilaginous cells, elastic cells, fibrous cells,myocytes, myocardial cells, endothelial cells, smooth muscle cells, andhematopoietic cells.

As used herein, the phrase “ex vivo” refers to living cells which arederived from an organism and are growing (or cultured) outside of theliving organism, preferably, outside the body of a vertebrate, a mammal,or human being. For example, cells which are derived from a human beingsuch as human muscle cells or human aortic endothelial cells and arecultured outside of the body are referred to as cells which are culturedex vivo.

Ex vivo induction of tissue formation can be effected, according to someembodiments of the present invention, by seeding the hybrid hydrogel asdescribed herein, or a composition-of-matter as described herein, withcells. Exemplary cells are as described hereinabove.

The phrase “tissue” refers to part of an organism consisting of anaggregate of cells having a similar structure and function. Examplesinclude, but are not limited to, brain tissue, retina, skin tissue,hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue,blood tissue, muscle tissue, cardiac tissue brain tissue, vasculartissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietictissue and fat tissue. Preferably, the phrase “tissue” as used hereinalso encompasses the phrase “organ” which refers to a fullydifferentiated structural and functional unit in an animal that isspecialized for some particular function. Non-limiting examples oforgans include head, brain, eye, leg, hand, heart, liver kidney, lung,pancreas, ovary, testis, and stomach.

The term “seeding” refers to encapsulating, entrapping, plating, placingand/or dropping cells into the hybrid hydrogel (orcomposition-of-matter).

It will be appreciated that seeding the hybrid hydrogel with cells canbe performed following the formation of the hydrogel or prior tohydrogel formation, i.e., by mixing the cells with the aqueous solutioncontaining the peptides and the polymer, as described hereinabove forgenerating the hydrogel. The concentration of cells to be seeded on thehydrogels depends on the cell type and the hydrogel properties.

In some embodiments, following seeding the cells on the hydrogel, thecells are cultured in the presence of tissue culture medium and growthfactors.

Thus, the hybrid hydrogels described herein can be formed in vitro, exvivo or in vivo, and can be used to induce tissue formation and/orregeneration and thus treat individuals suffering from tissue damage orloss.

Thus, according to another aspect of some embodiments of the presentinvention there is provided a method of repairing a damaged tissue in asubject in need thereto. The method can be regarded as a method oftreating a subject having a disorder characterized by tissue damage orloss.

Accordingly, the hybrid hydrogels described herein,compositions-of-matter, compositions, kits or articles containing thesame, can be used for repairing a damaged tissue in a subject of fortreating a subject having a disorder characterized by tissue damage orloss.

As used herein the phrase “disorder characterized by tissue damage orloss” refers to any disorder, disease or condition exhibiting a tissuedamage (i.e., non-functioning tissue, cancerous or pre-cancerous tissue,broken tissue, fractured tissue, fibrotic tissue, or ischemic tissue) ora tissue loss (e.g., following a trauma, an infectious disease, agenetic disease, and the like) which require tissue regeneration formedical or aesthetical purposes. Examples for disorders or conditionsrequiring tissue regeneration include, but are not limited to, livercirrhosis such as in hepatitis C patients (liver), Type-1 diabetes(pancreas), cystic fibrosis (lung, liver, pancreas), bone cancer (bone),burn and wound repair (skin), age related macular degeneration (retina),myocardial infarction, myocardial repair, CNS lesions (myelin),articular cartilage defects (chondrocytes), bladder degeneration,intestinal degeneration, and the like.

The phrase “treating” refers to inhibiting or arresting the developmentof a disease, disorder or condition and/or causing the reduction,remission, or regression of a disease, disorder or condition in anindividual suffering from, or diagnosed with, the disease, disorder orcondition. Those of skill in the art will be aware of variousmethodologies and assays which can be used to assess the development ofa disease, disorder or condition, and similarly, various methodologiesand assays which can be used to assess the reduction, remission orregression of a disease, disorder or condition.

The method is effected by contacting the damaged tissue or an organhaving tissue loss with the hydrogel as described herein or with acomposition-of-matter as described herein or with a pharmaceutical,cosmetic or cosmeceutical composition, as described herein. Contactingcan be effected by administering the hydrogel, composition-of-matter orcomposition to subject, preferably locally administering to the desiredsite of application (e.g., damaged tissue or organ with tissue loss), asdescribed herein. Optionally, contacting is effected by contacting thedesired site of application with the plurality of peptides and thebiocompatible polymer, as described herein.

It will be appreciated that whenever a composition-of-matter asdescribed herein comprises cells, the cells can be derived from thetreated individual (autologous source) or from allogeneic sources suchas embryonic stem cells which are not expected to induce an immunogenicreaction.

In some embodiments, treating the damaged tissue is effected by fillingthe gap of the defect by the hybrid hydrogel or composition as describedherein, so as to initiate the regeneration of new cartilage tissue.

As noted hereinabove, in some embodiments, the methods and uses asdescribed herein can be utilized for cosmetic or cosmeceuticalapplications, for example, for treating age-related damaged skin tissues(e.g., wrinkles), or trauma-related damages tissues, or for firming anyother defected skin area, nail area or defected mucosal tissue.

It is expected that during the life of a patent maturing from thisapplication many relevant biocompatible polymers and self-assembledpeptides will be developed and the scope of the terms “biocompatiblepolymer” and “self-assembled peptide” is intended to include all suchnew technologies a priori.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

The word “exemplary” is used herein to mean “serving as an example,instance or illustration”. Any embodiment described as “exemplary” isnot necessarily to be construed as preferred or advantageous over otherembodiments and/or to exclude the incorporation of features from otherembodiments.

The word “optionally” is used herein to mean “is provided in someembodiments and not provided in other embodiments”. Any particularembodiment of the invention may include a plurality of “optional”features unless such features conflict.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Example 1 Hybrid Hydrogel Formation and Characterization Materials andExperimental Methods

Materials:

Lyophilized Fmoc-diphenylalanine peptide was purchased from Bachem(Budendorf, Switzerland).

High molecular weight (3×10⁶) hyaluronic acid as a sodium salt wasobtained from BTG-Ferring (Kyriat Malahy, Israel) in syringes as asolution of 1% HA in PBS.

Formation of Hybrid Fmoc-FF/HA Hydrogels:

In a typical procedure, lyophilized Fmoc-FF peptide was dissolved inDMSO (Dimethyl sulfoxide) to achieve a concentration of 0.1 mg/ml. Thepeptide stock solution was diluted by adding HA (1%) solubilized inddH₂O (DDW), to achieve a final concentration 0.5% w/w (5 mg/ml) of thecombined substances. The mixture was physically blended by vortex andthen maintained at room temperature until gelation is complete, unlessotherwise indicated.

Different hybrid hydrogels were prepared by using different peptide-HAratios. Thus, a 25/75 w/w hydrogel was composed of 1.25 mg Fmoc-FF and3.75 mg HA in 1 ml solution. A 50/50 w/w hydrogel was composed of 2.5 mgFmoc-FF and 2.5 mg HA in 1 ml solution. A 75/25 w/w hydrogel wascomposed of 3.75 mg Fmoc-FF and 1.25 mg HA in 1 ml solution.

To avoid any pre-aggregation and pre-assembly, fresh peptide stocksolutions were prepared for each experiment.

Electronic Microscopy Measurements:

Three different electron microscopes have been used: TEM, SEM, andE-SEM, in order to cross-check and fully understand the morphologicalcharacterization of the newly formed hydrogels.

For TEM analysis, 100 μl samples of hydrogel were prepared and a portionof a sample was placed on a 400-mesh cupper grid. After 1 minute, thepiece of gel was removed, as well as excess of fluid. Negative stainingwas obtained by covering the grid with 10 μl of 2% uranyl acetate inwater. After 2 minutes, excess uranyl acetate solution was removed.Samples were viewed using a JEOL 1200EX electron microscope, operatingat 80 kV.

For SEM analysis, fresh pieces of formed gel were placed on microscopeglass cover slips and dried at room temperature, then spattered withgold. Images were obtained with a JSM JEOL 6300 scanning electronmicroscope operating at 5.0 kV.

E-SEM samples were prepared by placing pieces of gel on a microscopemetal stand. Images were obtained using an FEI QUANTA 200 E-scanningelectron microscope operating at 15.0 kV and with an FEI XL 30E-scanning electron microscope field-emission gun at 5.0 kV and underreduced pressure of 2.5 Torr (1 Torr≈133 Pa).

Density:

Upon hydrogel formation, a pre-determined volume was collected by tips,and weighed, for specific density, calculating gram/cm³. Hydrogels whichwere too rigid to be collected with a tip were cut with a scalpel todefined geometric shapes and the volume was calculated upon measuringthe dimensions of these shapes.

Swelling:

Identical volumes of hydrogel samples were placed on plates, weights(Wi) were recorded, and the hydrogels were then placed in ddH₂O. Toallow equilibration and swelling, all samples were allowed to swell for24 hours. In 4 different time intervals through 2 weeks, theequilibrated swollen mass (Ws) have been recorded by gently absorbingexcess of water from each sample. The hydrogel samples have beensubsequently lyophilized and their dry weights (Wd) measured. Theequilibrated swelling ratio (Q) was defined as the ratio of Ws to Wd.

Viscosity:

The apparent viscosity measurements of HA, Fmoc-FF and their hybridswere carried out using AR-G2 parallel plates rheometer (TA Instruments).Tests were carried out with a 20mm plate at 25° C. and 37° C., applyinga constant gap size of 1 mm Viscosity was measured using a stepped flowstep program, at increasing shear rates, to evaluate non-Newtonianbehavior of the hydrogels.

In addition, recovery after shear of the hydrogels was measured bycomparing the viscosity at t₀ (which has undergone the above elaboratedviscosity measurement test) to the viscosity of the same hydrogel after10 minutes rest (t₁₀) at the same temperatures.

Rheological Studies:

The in-situ hydrogel formation, mechanical properties, and kinetics werecharacterized by an AR-G2 rheometer (TA Instruments). Time-sweeposcillatory tests in parallel-plate geometry were performed on 210 μl offresh solution (resulting in a gap size of 0.6 mm) at room temperature.Oscillatory strain (0.01-100%) and frequency sweeps (0.01-100 Hz) wereconducted in order to find the linear viscoelastic region, at which thetime sweep oscillatory tests were performed. G′, which represents thegel stiffness, was obtained at 10 Hz oscillation and 1% straindeformation for each sample was used to compare the relative mechanicalstiffness of the hydrogels.

Results

Morphology Characterization:

The new hybrids include a natural component of HA and a syntheticcomponent of Fmoc-FF.

FIG. 2 presents a photograph taken by a digital camera of hydrogels madeof pure components and of various Fmoc-FF/HA weight ratios. As can beseen in FIG. 2, the macrostructure of all the different hydrogel hybridsexhibit a homogenous and transparent appearance. Upon comparisonsbetween the different hybrids, it can be seen that in hydrogel withhigher concentration of the peptide, the mechanical features are morepronounced and the hydrogels exert more brittle features, whereby inhydrogels made with higher concentration of HA, the hydrogel possesses asoft like fabric with a viscous behavior.

Under a centrifugation process, no segregation or phase separationoccurs in any of the hydrogel preparations, suggesting that theseconstructs are homogenous biomaterials, well mixed, and blended.Moreover, two of the three hybrids (25/75, 50/50) were found suitablefor injection through a thin needle, gyge of 21.

To gain more insight regarding the molecular organization of theself-assembled structures of Fmoc-FF and HA, TEM, SEM and E-SEM analyseswere performed. The results, presented in FIGS. 3A-C, show that allhybrid hydrogels have a nature of fibrous networks (FIGS. 3A-C; (2), (3)and (4)), whereby higher concentrations of Fmoc-FF resulted in higherformation of tubular structures, and more dense structures. Hydrogelmade of Fmoc-FF exhibited self-assembled tubular form (FIGS. 3A-C; (5)).

While all of the peptide-containing hydrogels form open-ended tubularstructures, a remarkable size uniformity of the tubular structures'diameter was observed for all hydrogel's types, with the diameter beingabout 30-40 nm. However, the length of the tubular structures differsbetween the hydrogels. The hybrids Fmoc-FF/HA 25/75 and 50/50 havesimilar lengths ranging between 21±3 μm and 22±3 μm, respectively. TheFmoc-FF control hydrogel has a shorter length of 18±2 μm, while thehybrid Fmoc-FF/HA 75/25 has a surprisingly shorter length of only 11±2μm.

It can be seen from the results that the hybrids Fmoc-FF/HA 25/75 and50/50 are very much alike in their appearance and behavior, while thehybrid Fmoc-FF/HA 75/25 exhibits features that are more similar to theFmoc-FF.

The E-SEM images (FIG. 3C (2), (3) and (4)) show a crystallized coatingon top of the peptide nanotubes, seen as the brighter sections in theseimages. The crystallized coating is presumably formed of salts presentin the PBS solution used for forming the hybrid hydrogels.

The time lapse to the crystallization appearance depends on theFmoc-FF/HA ratio. In the 25/75 hybrid, crystallization occurs after 20minutes at 5 Torr pressure and down to 3 Torr. In the 50/50 hybrid, ittakes around 35 minutes under the same conditions. In the 75/25 hybrid,which is a denser hydrogel with many nanotubes and less HA, thecrystallization lasts longer than 45 minutes. These data may suggestthat HA is caged within the peptidic structure, such that in denserstructures, with less HA, less crystallization is observed.

Density:

FIG. 4 presents the values calculated for the density of the varioushydrogels obtained. As can be seen in FIG. 4, density measurementsfurther suggest that the hybrid hydrogel made from 75/25 Fmoc-FF/HA hasdifferent features than the other tested hybrids (25/75 and 50/50Fmoc-FF/HA).

Without being bound to any particular theory, it is assumed thatcombining HA with Fmoc-FF results in moderate hybrids that have averagedfeatures. The original HA solution and Fmoc-FF have a similar density ofabout 1.012 and 1.022 gram/cm³, respectively, which is close to the DDWand PBS density (1.016 g/cm³). The hybrids 25/75 and 50/50 Fmoc-FF/HAhave lower densities of 0.972 and 0.987 g/cm³, respectively, which canbe explained by the interference of the viscous HA molecules within thecreation and organization of the Fmoc-FF nanotubes. It is to be notedthat both HA and Fmoc-FF are negatively charged in aqueous solution atpH 7, and thus may experience electrostatic repulsion therebetween. Inaddition, HA molecules are hydrated, and thus present a hydrophilicnature, while the Fmoc-FF has a hydrophobic nature, due to its aromaticrings.

Along this line, the more Fmoc-FF is in the hydrogel, the denser thestructure should be, as is indeed shown in FIG. 4.

The length of the formed tubular structures may also affect the finaldensity, as shown for the two hybrids hydrogels made of 25/75 and 50/50Fmoc-FF/HA, which were found to have the longest structures, asdiscussed hereinabove, and lower densities. Longer tubes are assumed tocause a less compact organization. The Fmoc-FF hydrogel has shorternanotubes, resulting in a denser hydrogel.

This may also explain the higher density of the 75/25 Fmoc-FF/HA hybridhydrogel, which was found to have the shortest nanotubes, as describedhereinabove.

Because all hydrogels have the same dry weight (5 mg/ml), densityrepresents the average pore size within the network, the pore sizedistribution, and the pore interconnections, which are features of ahydrogel matrix. These features of the porous structure determine theabsorption (or partitioning) and diffusion of solutes through thehydrogel [A. S. Hoffman 2002 Advanced Drug Delivery Reviews 54(1):3-12].

Swelling:

The character of the water in a hydrogel can determine the overallpermeation of nutrients into, and cellular products out of, the gel[Hoffman 2002, supra]. The swelling ratio of the initial state of all ofthe hydrogels can be calculated from the hydrogel concentration used,0.5%, which means 5 mg dry weight (Wd) in a total of 1000 μl (or 1000mg) swelling weight (Ws). Dividing Ws by Wd resulted in a swelling ratioof 200 at the initial state (see, FIG. 5).

HA has a swelling capacity of 1000, as taken from the literature[Balazs, E. A. In Chemistry and biology of hyaluronan; Garg, H. G., C.A. Hales, C. A., Eds.; Elsevier ltd. 2004, Chapter 20], which representsits high hydration rate. The open, random coil structure of hyaluronicacid exhibits large solvent domains due to the large number ofhydrophilic residues. This gives hyaluronic acid, even at lowconcentrations, the character of a high viscosity solution, which isresponsible for its outstanding lubrication properties. However, thisfact is of a great disadvantage as HA immediately disappears in aqueousenvironment and does not retain its structure.

As shown in FIG. 5, the Fmoc-FF hydrogel has a swelling ratio of 212,similar to the initial state, which can be explained by its hydrophobicnature and hence its poor hydration ability.

As further shown in FIG. 5, all hybrid Fmoc-FF/HA hydrogels retainedtheir structure even after 2 weeks in aqueous environment, and exhibiteda swelling ratio averaging between the Fmoc-FF hydrogel and the HA. Itcan be seen that the higher the concentration of the peptide, the lessswelling is attained.

It is noted that the hybrid hydrogel made of 75/25 Fmoc-FF/HA, whichrepresents a denser state than the Fmoc-FF hydrogel, as discussedhereinabove, has a swelling ratio higher than that of the Fmoc-FFhydrogel (224 and 212, respectively). In addition, it is noted thatalthough HA solution and Fmoc-FF hydrogel have almost the same density,their swelling properties are very diverse. Without being bound to anyparticular theory, it is suggested that the swelling ratio is affectedby the elasticity of the chains composing the hydrogel. HA chains arevery elastic and flexible, and can be pushed by water (and cells) whilepassing thereby. However, Fmoc-FF forms a dense network of tubularnanostructures through which water passing is limited. It is thereforesuggested that the main factor affecting the swelling ratio is not thedensity of the solution, but the amount of the hydrophilic, flexiblechains, of the HA, which have a high hydration rate and can be pushed bymolecules passing therethrough.

Viscosity:

In order to explore the mechanical features of the hydrogels, theviscosity of the different hybrids was analyzed and compared to thecontrol hydrogels—HA and Fmoc-FF (FIG. 6). All gels show the samebehavior, where as the shear rate increases, the viscosity decreases.The viscosity was studied at 25° C. and 37° C. Very similar trends wereexhibited at 37° C., with a reduction in viscosity at 37° C. incomparison with the 25° C. values, as expected. The higher thetemperature, the solutions exhibit a more liquid nature and less flowresistance, expressing lower viscosity.

HA is known as a very viscoelastic solution [Coviello, T.; Matricardi,P.; Marianecci, C.; Alhaique, F. J Control. Rel. 2007, 119(1), 5-24]. Atlow shear rates the viscosity of a 1% HA solution is 150 Pa·s and itdecreases moderately with the increase in shear rate (FIG. 6). HA alsoshows a high viscosity recovery tendency, with almost no change inviscosity between the two time intervals tested (t₀ and t₁₀). The highviscosity recovery of HA is supported, for example, in FIG. 6, where itis shown that shear rates minimally affect the viscosity of HA.

Fmoc-FF hydrogel (0.5%) exhibits a very high viscosity value of morethan 5×10⁵ Pa·s at low shear rates, showing a sharp decrease withincreasing shear rates, due to the brittle nature of this hydrogel. Incontrast to HA, Fmoc-FF shows low recovery after shear, as it exhibitslower values of viscosity upon measurement after 10 minutes rest (FIG.7). The viscosity change between t₀ (viscosity at initial time) and t₁₀(viscosity after 10 minutes rest) is indicative of the elasticity of thesolution. Thus, the less change in the viscosity, the more elastic isthe hydrogel.

All three hybrids show average levels of viscosity, between HA andFmoc-FF. It can be seen that as the concentration of Fmoc-FF in thehydrogel hybrid is higher, the viscosity is higher (FIG. 6), and therecovery is lower (FIG. 7).

All three hybrids are composed of HA, which is a very viscoelasticsolution, and Fmoc-FF, which is a very rigid but brittle hydrogel,therefore not showing full recovery after shearing. The hybrids 25/75and 50/50 show very similar viscosity values with no significantdifference, which can be explained by the morphological features ofthese hydrogels, as discussed hereinabove. The hybrid 75/25 shows valueswhich resemble more the Fmoc-FF hydrogel, as can be also explained byits morphological features discussed supra.

In exemplary data obtained while measuring viscosity recovery aftershear, it was shown that at shear rate of 0.1 sec⁻¹ the viscosity of a50/50 Fmoc-FF/HA hybrid hydrogel was 573.7 Pa·s and after 10 minutes325.8 Pa·s, representing 56% recovery.

The viscosity of Fmoc-FF hydrogel at the same shear rate was 2,038 Pa·sand after 10 minutes only 68 Pa·s, representing 3.3% recovery. At ashear rate of 1 sec⁻¹, the 50/50 Fmoc-FF/HA has a viscosity at t=0 of23.43 Pa·s and at t=10 minutes 18 Pa·s, representing 76.8% recovery.Under the same shear rate, a Fmoc-FF hydrogel has at t=0 viscosity of29.83 Pa·s and at t=10 minutes 10.59 Pa·s, representing a recovery of35.5%.

Rheological Study:

The mechanical properties of the hydrogels can be used to determinetheir suitability to various applications. Hence, rheologicalcharacterization was conducted in order to study the kinetics of thehydrogel formation and the viscoelastic properties of the materials.

The complex shear modulus (G*) of the hydrogels showed that the elasticresponse component (G′, storage modulus) exceeded the viscous responsecomponent (G″, loss modulus, not shown) by at least an order ofmagnitude, indicating that a phase transition into a viscoelasticmaterial occurred.

As can be seen in FIGS. 8-10, the different hydrogels exhibiteddifferent G′ values and different kinetics of hydrogel formation.Rheological behavior was affected by both hydrogel's composition (FIG.8), hydrogel's concentration (FIG. 9), and temperature (FIG. 10), asdiscussed in further detail hereinunder.

The obtained data is summarized in Table 1 below.

TABLE 1 4° C. Hydrogel type G′ [Pa] 25° C. 37° C. & final after G′Gelation G′ Gelation concentration [%] 40 min [Pa] time [min] [Pa] time[min] HA 1 162 158 — 135 — FmocFF 0.5 963 8767 10.5 9564 8.3 FmocFF 1 —122700 13.5 60770 7 25/75 0.5 524 1158 78 1227 17 50/50 0.5 1474  207616.5 1951 5.5 50/50 1 — 5030 18 4658 8.3 75/25 0.5 14860  25730 5.519000 1.2 75/25 1 — 85700 6 52020 2.5

FIG. 8 presents the values obtained for the storage shear modulus (G′)as a function of time, for different hybrid hydrogels and for Fmoc-FFhydrogel at a concentration of 0.5% (5 mg/cc), and of an HA solution at1%. The gelation time is determined by the time to reach its plateau, orthe time the highest G′ is achieved, and is indicative of the timerequested for the tubular structures to arrange within the hydrogel. HA0.5% hydrogel demonstrates G″ values higher than G′ values, which aretypically indicative for liquid solutions. HA at 1% demonstrates G′ fourtimes higher than G″ (178 and 46 Pa, respectively). The measured G′value is very low when compared to the other tested hydrogels, and isindicative for the softness of HA. The Fmoc-FF hydrogel demonstrates avery rigid biomaterial, with a G′ of almost 9000 Pa and a gelation timeof 10 minutes.

The hybrids 25/75 and 50/50 Fmoc-FF/HA demonstrate an averaged G′, asexpected, with very similar values of around 1200 Pa. Interestingly,similar rigidity was observed for these two hydrogel types, althoughdifferent amounts of Fmoc-FF were used and thus the extent of tubularstructures formation is different. These two hybrid hydrogel differ isthe gelation time, which was longer in the 25/75 Fmoc-FF/HA hybrid.

The hybrid 75/25 Fmoc-FF/HA hydrogel was found to exhibit high rigidityof more than 25000 Pa and the lowest gelation time of only 5 minutes.These results are in accordance with the short nanotubes length of thishydrogel type, described hereinabove.

FIG. 9 presents the effect of hydrogel concentration (0.5% or 1%) on therigidity, as measured at 25 ° C. As can be seen in FIG. 9 and Table 1,all hydrogels exhibited similar kinetic pattern with lower G′ values asthe concentration decreased. When concentration decreases in half (from1% to 0.5%), G′ values decrease more than 3 times, while the gelationtime stays the same.

However, as can be seen in FIG. 10, various kinetic patterns wereobserved at different temperatures (for the same concentration of the50/50 Fmoc-FF/HA hybrid hydrogel, as a representative example). Theobtained data indicate that in hydrogels exhibiting lower G′ values(less than 5000 Pa) temperature change from 25° C. to 37° C. has lesssignificant effect on the G′ value, yet affects the gelation time,probably due to faster self-assembly at higher temperatures.

As shown in Table 1, more rigid hydrogels (G′ more than 5000 Pa) exhibitnot only a change in the gelation time, but also a significant change inthe G′ values as a result of temperature change. The more Fmoc-FF in thehydrogel, the more dramatically the elastic properties (G′) change athigh temperature.

The temperature effects can be explained by self-assembly velocities,whereby at higher temperature, faster self-assembly results in reducedformation of nanotubes.

Example 2 Degradation of Hybrid Gels

Hydrogels' biodegradation was examined in the presence of hyaluronidase,an enzyme that degrades hyaluronic acid. The hybrid hydrogels describedherein are designed so as to result in a slower degradation rate andlonger retention time in the body, compared to that of HA, yet withoutusing chemical cross-linking.

Assay Protocol:

The in vitro enzymatic degradations of the hydrogels were measured as afunction of time by incubating the gels in the presence of hyaluronidase(Hyase), by monitoring the residual mass of the hydrogel; and bydetermining the released glucuronic acid from the hydrogel to thesolution by a Dische assay, as described in [Z. Dische 1947 J. Biol.Chem. 167: 189-198]. In brief, 0.2 ml of 0.1% carbazole solution inalcohol was added to 1 ml of a tested sample. After vortexing, 6 ml ofconcentrated H₂SO₄ was added to the solution. The glass tubes wereclosed with coupled sleeves, boiled for 15 minutes in a water bath, andthe solution was then chilled to room temperature and the absorption(λ=527 nm, colored pink—violet) was measured relatively to the blankreference solution.

Bovine testicular hyaluronidase, Type IV-S, as lyophilized powder (451U/mg) was purchased from Sigma Aldrich (Rehovot, Israel).

One ml of each hydrogel was transferred into custom-made plastic tubeswith 40 μm filter paper placed on the bottom. The tubes with thehydrogels were placed in glasses soaked with buffer (0.1 M monosodiumphosphate in 0.15 M NaCl (pH 5.3)). Enzyme concentration of 10 U/mlbuffer was chosen to imitate by approximation the endogenous enzymelevels. Enzyme concentrations of 1.25 U/ml, 2.5 U/ml, 3.75 U/ml wereused for the 75/25, 50/50 and 25/75 Fmoc-FF/HA hydrogels, respectively,in order to match the amount of HA in the hydrogel to the enzymeconcentration.

The hyaluronic acid release is uniaxial, taking place only at theinterface of the hydrogel and buffer.

The gels were incubated for various time intervals at 37° C. with mildmixing on a platform shaker (50 rpm), for a week. At each time point,the solution was removed to determine the residual HA quantity byDische's assay, as described hereinabove, and the residual gels' masses.The tubes were then returned to their glasses and replenished with freshHyase buffer solution for the remaining residues. Pure hyaluronic acidsolution and Fmoc-FF hydrogel were used as a positive and negativecontrol, respectively.

Results:

FIG. 11 presents the hydrogel's mass loss during the assay period (oneweek) upon incubating the tested hydrogels in the presence or absence ofhyaluronidase. As shown in FIG. 11, substantially lower degradation wasobserved for hybrid hydrogels as compared to HA solution, withdegradation extending from a few hours for the HA solution to 7 days.The enzyme concentration used has not affected the degradation rate(data not shown).

The obtained results suggest that enzyme penetration through the hybridhydrogel network is limited, such that HA disappearance occurs as aresult of its migration out of the hybrid complex. These data are incorroboration with the results obtained in the swelling assay.

FIG. 12 presents the data obtained for the release of glucuronic acid(as a result of HA enzymatic degradation) into the tested solution,which are in corroboration with the mass loss data. The hybrid 50/50Fmoc-FF/HA hydrogel showed similar behavior in the presence and absenceof the enzyme, again probably due to the poor penetration ability of theenzyme into the hydrogel. The hybrid 25/75 Fmoc-FF/HA hydrogel, whichwas found to exhibit higher swelling ability, exhibits higher extent ofHA degradation.

Example 3 Biocompatability Assays

Cells Viability Test:

To verify the ability of the hybrid hydrogels described herein to beused in biological applications, their biocompatibility was determinedusing an in vitro cell culture experiment. Three types of cells werecultivated on top of the hybrid hydrogels: CHO (Chinese HamsterOvaries), fibroblasts and chondrocytes.

Assay Protocol:

CHO, fibroblasts and chondrocytes cells were grown in Dulbecco'sModified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 100U/ml penicillin, 100 U/ml streptomycin, and 2 mmol/liter L-glutamine(all from Beit Haemeck, Israel). Cells were maintained at 37° C. in ahumidified atmosphere containing 5% CO₂. Subconfluent cultures wereharvested by trypsinization, counted, and diluted in specific cells'media to form a cell concentration of 10⁶ cells/ml. The gels swelledovernight with 0.1 ml DMEM. The next day, DMEM was removed and 0.1 μl ofthe CHO cells (10⁵ cells) were placed on top of the hydrogel. After 1,3, and 7 days of incubation at 37° C. the viability of the cells wasdetermined using an MTT assay.

Results:

The cell viability was analyzed 1, 3, and 7 days post seeding. Cellsseeded as monolayers with PBS were used as control. Cell viability wasanalyzed using an MTT assay. When MTT was added to the cell culturemedium, the mitochondrial dehydrogenase enzyme, which is present only inlive cells, changed the color of the yellow MTT to dark-blue crystals,which accumulated inside the living cells and give a clear indication ofcell viability.

As can be seen in FIG. 13, after 1 day, the cultured CHO cells showedhigh viability on both HA and the hybrid hydrogels. Cells on Fmoc-FFhydrogel showed lower viability of 50%. The viability of CHO cells onall hybrid hydrogels increased with time, whereas a moderated decreasein cell viability was recorded for the Fmoc-FF hydrogel.

In the other cell types tested, normal human fibroblasts andchondrocytes, a viability of about 80% was measured on the first day,for all hybrid hydrogels (data not shown). Without being bound to anyparticular theory, it is assumed that since these cells are primarycells, their sensitivity is higher than that of the CHO cell line. It isfurther assumed that the viability of primary cells is influenced fromthe Fmoc-FF/HA ratio in the hydrogel. As discussed hereinabove, it isassumed that the results are influenced by the permeation properties ofthe hydrogel, which limit the diffusion rate of the medium through thehydrogel, and may also affect the diffusion of the MTT reagent.

FIG. 14 represents an image of chondrocytes seeded on the hybrid 50/50Fmoc-FF/HA hydrogel, showing that cell survived, and that theircharacteristic polygonal phenotype shape was captured.

Cell Dispersion:

Assay Protocol:

After mixing the components forming the hydrogel hybrids (immediatelyafter vortex), 50 μl of each mixture was placed in a different well in a96-well plate. HA solution and Fmoc-FF hydrogel were used as controls.Once gel formation was observed (for hybrid hydrogels and Fmoc-FF),100μl of DMEM was added to each well and the plate was placed in anincubator for overnight. Then, the medium was removed and 2.5×10⁴ MSC(mesenchymal stem cells) ATCC in 100 μl fresh medium was seeded on topof each solution or hydrogel. Additional 100 μl DMEM was added, and theplate was incubated. After 2, 6 and 10 days of incubation, cell quantitywas measured with a hemocytometer. A distinction between cells grown onthe bulk gel or on the plate underneath was made by separatelycollecting medium & gel and then collecting the cells which grow on theplate by trypsinization.

Results:

FIGS. 15A-B present the data obtained for percents of cells found inmedium and gel out of the total cell amount at different time points(FIG. 15A), and for percents of cells found grown on the plate out ofthe total cell amount at different time points.

It can be seen that cell proliferation in HA solution is very similar toplain plate. Even after 2 days, almost all the cells slipped down andgrown on the plate. On the other hand, with the hydrogel hybrids, thecells remained in the gel even after 10 days. These remarkable resultsdemonstrate the capability of the hybrid hydrogels to serve as anefficient scaffold for cells, a capability imparted by the enhancedrigidity of the hybrid hydrogels as compared with HA viscous solutions.

Example 4 In ovo CAM Assay

Assay protocol:

Chicken eggs in the early phase of breeding are between in vitro and invivo systems and provide a vascular test environment to study toxicityof biomaterials, especially hydrogels. After the chick chorioallantoicmembrane (CAM) has developed, its blood vessel network can be easilyaccessed, manipulated and observed and therefore provides an optimalsetting for allo- and xenografts.

For experimental purposes, the tested substances are placed on thevasculature network of capillaries of the CAM of fertilized chickeneggs, upon 8 days in gestation. Detailed methods of CAM assays aredescribed in Dohle et al. J Vis Exp. 2009 Nov 30;(33).

In brief, exogenous limb-buds of a 4-day fertilized chicken egg wereadded to the tested Fmoc-FF/HA hybrid hydrogels. In embryology, limb budis a structure formed by the developing limb. At day 4-4.5 to gestation(stage 24), the limb bud is full of MSCs (mesenchymal stem cells), whichare multipotent stem cells that can differentiate into a variety of celltypes, including: osteoblasts (bone cells), chondrocytes (cartilagecells) and adipocytes (fat cells).

The limb bud embedded in the hydrogel was placed on an 8-day fertilizedegg (CAM), and after 6 days, the obtained tissue was removed andsubjected to histology evaluation.

Results:

FIGS. 16A-C present light micrographs of H&E staining of limb-budsembedded in 50/50 Fmoc-FF/HA hybrid hydrogel after 6 days on a CAM of an8-day fertilized chicken egg (X 25). FIG. 16A presents an initial stageof limb-bud; FIG. 16B presents chondrocytes condensation; and FIG. 16Cpresents organogenesis into limb formation.

As can be seen, the 50/50 hybrid hydrogel can mimic the stages ofdevelopmental limb, as occurs in embryology: first is the chondrocytecondensation into islands, as can be seen in FIG. 16B, and later is thelimb formation (FIG. 16C). The MSCs not only survive on the 50/50hydrogel, but also proliferate and differentiate into a limb, thusdemonstrating the improved and potent biocompatibility of the hybridhydrogels described herein.

Example 5

Formation of Hydrogel Hybrids Made of Chitosan and Self-AssemblingPeptides

Materials and Methods

Chitosan having MW of 64000Da was obtained from Koyo chemical CO., Ltd.,Japan.

A 2% solution (20 mg/ml) of chitosan in acetic acid (0.25N) was preparedand after one day was titrated with NaOH 0.1N in order to achieve pH6.9. A 0.1 mg/ml solution of Fmoc-FF in DMSO was thereafter added to thechitosan solution, the mixture was vortexed and maintained at roomtemperature until gel formation was observed. The amount of Fmoc-FF inthe final mixture was 0.5 mg or 2 mg.

Thus, 2 μl or 5 μl of the peptide stock solution (0.2 mg or 0.5 mgFmoc-FF, respectively) was added to the chitosan solution to afford asolution containing and 22 mg/2020 μl (˜2.2% hydrogel) or 20.5 mg/2005μl (2.05% hydrogel), respectively.

Results:

Hydrogel formation was observed within a few minutes. The formedhydrogels exhibit homogenous and transparent appearance, with a higherrigidity than the regular chitosan solution. Hydrogels containing thehigher Fmoc-FF concentration were observed as having higher mechanicalstrength and more brittleness. Under a centrifugation process (10000 rpmfor 20 seconds), no segregation or phase separation occurred in any ofthe formed hydrogels, indicating the stable nature of the hydrogelnetwork.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A hydrogel comprising a fibrous network of aplurality of peptides and at least one biocompatible polymer, whereinsaid peptides are capable of self-assembling in an aqueous solution soas to form a hydrogel and wherein said biocompatible polymer features atleast one characteristic selected from the group consisting of: (i) astorage modulus G′ lower than 500 Pa at 10 Hz frequency and at 25° C.;(ii) a swelling ratio (Q) higher than 500; (iii) a viscosity at 0.1Sec⁻¹ shear rate and at 25° C., lower than 300 Pa·s; and (iv) aviscosity recovery after shear of at least 95%.
 2. The hydrogel of claim1, wherein said biocompatible polymer is a polysaccharide.
 3. Thehydrogel of claim 1, wherein each peptide in said plurality of peptidesis a dipeptide.
 4. The hydrogel of claim 3, wherein each of saiddipeptides is a homopeptide.
 5. The hydrogel of claim 1, wherein eachpeptide in said plurality of peptides is an end-capping modifiedpeptide.
 6. The hydrogel of claim 5, wherein said end capping modifiedpeptide comprises at least one end capping moiety, said end cappingmoiety being selected from the group consisting of an aromatic endcapping moiety and a non-aromatic end-capping moiety.
 7. The hydrogel ofclaim 6, wherein said aromatic end capping moiety is9-fluorenylmethyloxycarbonyl (Fmoc).
 8. The hydrogel of claim 1, whereina total concentration of said plurality of peptides and said polymerranges from 0.1 weight percent to 5 weight percents of the total weightof the gel.
 9. The hydrogel of claim 1, wherein a weight ratio of saidplurality of peptides and said polymer ranges from 10:1 to 1:10.
 10. Thehydrogel of claim 1, characterized by at least one of: a storage modulusG′ to loss modulus G′ ratio that is greater than 4; a storage modulus G′higher than 1,000 Pa at 10 Hz frequency and at 25° C.; and a storagemodulus G′ lower than 100,000 Pa at 10 Hz frequency and at 25° C. 11.The hydrogel of claim 1, characterized by: a viscosity that ranges from200 to 2000 Pa·s at 0.1 Sec⁻¹ shear rate, at 25° C.; a viscosityrecovery after shear of at least 50%, at 0.1 sec⁻¹; and a swelling ratio(Q) that ranges from 100 to
 500. 12. A composition-of-matter comprisingthe hydrogel of claim 1 and at least one agent being incorporatedtherein or thereon.
 13. A process of preparing the hydrogel of claim 1,the process comprising contacting said plurality of peptides and saidbiocompatible polymer in an aqueous solution.
 14. A pharmaceutical,cosmetic or cosmeceutical composition comprising the hydrogel ofclaim
 1. 15. A pharmaceutical, cosmetic or cosmeceutical compositioncomprising the composition-of-matter of claim
 12. 16. Anarticle-of-manufacture comprising the hydrogel of claim
 1. 17. Anarticle-of-manufacture comprising the composition-of-matter of claim 12.18. A kit for forming the hydrogel of claim 1, the kit comprising saidplurality of peptides and said biocompatible polymer.
 19. The kit ofclaim 18, further comprising an aqueous solution, wherein said peptidesand said aqueous solution are individually packaged within the kit. 20.A kit of for forming the composition-of-matter of claim 12, the kitcomprising said plurality of peptides, said biocompatible polymer andsaid active agent.
 21. The kit of claim 20, further comprising anaqueous solution, wherein said plurality of peptides and said aqueoussolution are individually packaged within the kit.
 22. A method ofrepairing a damaged tissue, the method comprising contacting the damagedtissue with the hydrogel of claim
 1. 23. A method of repairing a damagedtissue, the method comprising contacting the damaged tissue with thecomposition-of-matter of claim 12.